Studies on the Binding Specificity of Intelectin by Amira Ibrahim Aly

307 Pages·2014·7.29 MB·English

Studies on the Binding Specificity of Intelectin

by

Amira Ibrahim Aly Khalil

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Department of Chemistry
University of Alberta

© Amira Ibrahim Aly Khalil, 2015
Abstract
Our primary goal of this study was to obtain a better understanding for the
carbohydrates binding interactions with human intelectin (hIntL). Studying the
binding specificity of hIntL could help in discovering its natural substrate and
might give us a clue about its role in the innate immune response as well as its
pathophysiological action in various diseases.
In this project, recombinant expression of intelectins was successfully
done using HeLa cells and H5V cells. Yeast (Pachia pastoris) did not proove to
be a promising expression system for hIntL. Using chemical synthesis,
compounds (3.1–3.9) were obtained to be screened as ligands for hIntL. Herein,
we used surface plasmon resonance (SPR) as our analytical tool to study hIntL
binding specificity towards various synthesized Galf-containing oligosaccharides.
Staudinger ligation chemistry as well as amine coupling were used to immobilize
the carbohydrate epitopes to the SPR biosensor surface. Both hIntL-1 and hIntL-2
were screened against the derivatized surface.

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To the soul of my father.
To my mother.
To Amgad, Omar and Habiba.

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Acknowledgement
I want to show my gratitude to many people who supported me to achieve
my goal and helped me throughout my Ph.D. First I want to thank my supervisor
Professor Dr. Todd Lowary for being always supportive and helpful. He guided
me a lot during my project through his suggestions and advices. I was really
fortunate to be a member in his research group in the past five years.
I want to thank my supervisory committee; Professor Drs. West and
Campbell for their support and guidance. Many thanks goes also to Dr.
Christopher Cairo for his great help and advices during the binding studies using
surface plasmon resonance.
I should not forget also to show my abberiation to Ruixiang Blake, Chun
Xia Zou and Gareth Lambkin for teaching me many biological and molecular
biology techniques and methods. I want to thank our collaborators in complex
carbohydrate research center (University of Georgia, USA) for providing us with
the cell line for the protein expression.
I can’t forget to thank Dr. Jing Li (a former Ph.D. student in Lowary
group). She was very supportive and encouraging when I first started my Ph.D
and joined the group. Many thanks to all Lowary group members including
former members (Dr. Myles Poulin, Dr. Hashem Taha, and Dr. Michele
Richards).
I want also to thank Dr. Maju Joe for his chemistry tips and advices. Dr. Joe was
always there to help, advice, and support. I really learned a lot from his
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experience as an excellent chemist. Thanks to my friends Claude, Anushka and
Dr. Kamar.
I could not have my dream came true without the support I had from my
family and friends. Thanks my husband Dr. Amgad Albohy for being always nice,
kind, caring, supportive and most of all a great father. Many thanks to my kids
Omar and Habiba for their patience and smiles which gave me the energy and
motivation to fulfill my goal.
Finally, I want to express my deep appreciation and gratitude to my
mother who always meant everything for me. Thanks a lot my mother for your
care, advices, support and encouraging. I wish I could be a great mother like my
mom. Thanks to all my friends, family members and colleagues, I wish I could
mention all the names here.

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Table of contents

1. Introduction ................................................................................................... 1
1.1. Galactofuranose occurrence in natural oligosaccharide structures ......... 2
1.1.1. Mycobacteria ..................................................................................... 3
1.1.2. Bacteria ............................................................................................. 4
1.1.3. Protozoa ............................................................................................ 6
1.1.4. Fungi ................................................................................................. 6
1.2. Galactose-binding lectins ........................................................................ 8
1.2.1. Galectins ........................................................................................... 9
1.2.2. Biological function of the dendritic cell-specific intercellular
adhesion molecule-3-grabbing non-integrin (DC-SIGN) ............................. 11
1.2.3. Discovery of human intelectins....................................................... 14
1.3. Clinical significance of hIntL ............................................................... 32
1.3.1. Infection .......................................................................................... 32
1.3.2. Cancer ............................................................................................. 33
1.4. Project objectives .................................................................................. 35
1.5. References ............................................................................................. 37

2. Expression and Characterization of Intelectins ....................................... 58
2.1. Introduction ............................................................................................ 59
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2.1.1. Recombinant protein expression systems ....................................... 59
2.2. Results and discussion ............................................................................ 60
2.2.1. Attempts to express intelectin in Pichia pastoris ........................... 60
2.2.2. Intelectin expression in HeLa cells ................................................. 67
2.2.3. Intelectin expression in mouse endothelial H5V cell line............... 69
2.2.4. Characterization of hIntL-1 and hIntL-2 ......................................... 69
2.3. Experimental procedures ........................................................................ 71
2.3.1. Preparation of pPIC9/hIntL construct ............................................. 71
2.3.2. mIntL-2 expression in HeLa cells ................................................... 71
2.3.2.1 Maintenance of HeLa cell line .......................................................... 74
2.3.3. Intelectin expression in mouse endothelial cells (H5V) ................. 77
ii) hIntL-1 expression .................................................................................... 77
iii) hIntL-1 purification using affinity sepharose 6B column ....................... 78
2.4. Conclusion .............................................................................................. 81
2.5. References .............................................................................................. 83
3. Synthesis of the target oligosaccharides .................................................... 89
3.1. Introduction ............................................................................................ 90
3.1.1. General methods used to obtain Galf derivatives............................ 91
3.2. Synthetic strategies for the synthesis of the target compound (3.1–3.9).96
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3.2.1. Synthetic strategy for oligosaccharides containing β-D-Galf-(1→5)-
β-D-Galf and β-D-Galf-(1→6)-β-D-Galf linkages (3.1–3.4) ........................ 97
3.2.2. Synthesis of compound containing β-D-Galf-(1→6)-β-D-Glcp (3.7)
and β-D-Galf-(1→6)-α-D-Manp linkages (3.8) .......................................... 116
3.2.3. Rationale for the synthesis of compound 3.9 containing α-L-Araf 119
3.3. Conclusion ............................................................................................ 123
3.4. General methods ................................................................................... 123
3.4.1. General procedure for the deprotection of benzoylated products
using Zémplen conditions ........................................................................... 124
3.4.2. General procedure for the removal of levulinoyl protecting groups
……………………………………………………………………125
3.5. Experimental procedures ...................................................................... 125
3.6. References ............................................................................................ 178
4. Binding specificity of intelectins using surface plasmon resonance ..... 187
4.1. Introduction .......................................................................................... 188
4.2. Results and discussion .......................................................................... 191
4.2.1. Choice of the biosensor chip and surface functionalization method
……………………………………………………………………191
4.2.2. Staudinger ligation ........................................................................ 193
4.2.3. Synthesis of NHS-activated phosphine reagent 4.8 ...................... 196
4.2.4. Lectin binding ............................................................................... 198
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4.2.5. hIntL-1 binding data from Consortium for Functional Glycomics
(CFG) ……………………………………………………………………212
4.2.6. Conclusion .................................................................................... 216
4.3. General Methods .................................................................................. 218
4.3.1. Synthetic procedures ..................................................................... 218
4.3.2. Biosensor surface preparation using amine coupling ................... 224
4.3.3. Biosensor surface preparation using Staudinger ligation .............. 225
4.3.4. Detection of lectin binding specificity .......................................... 225
4.3.5. Competitive binding experiment with hIntL-1 using SPR............ 226
4.3.6. Surface regeneration ..................................................................... 227
4.3.7. Fluoresecnt labelling of hIntL-1 ................................................... 227
4.3.8. hIntL-1 screening against pathogen glycan array through CFG ... 229
4.4. References…………………………………………………………..…..230
5. Summary and future directions ............................................................... 240
5.1. Summary .............................................................................................. 241
5.2. Future directions ................................................................................... 244
5.2.1. Completion of the binding experiments ........................................ 244
5.2.2. Studying the binding interactions using other analytical methods 244
5.2.3. Expansion of the carbohydrate library and synthesis of more ligands
……………………………………………………………………245
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5.2.4. Study structure–activity relationships (SAR) between ligands with
high affinity and intelectins ........................................................................ 246
5.2.5. Conducting in vitro assays with live bacteria ............................... 247
5.3. References…………………………………………………………..…..248
6. Appendix .................................................................................................... 250
6.1. SPR sensograms for different lectins. .................................................. 251
Bibliography…………………………………………………………………….258

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