The Development of a Diagnostic Test for Heparanase by sif19147

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									 The Development of a Diagnostic Test
           for Heparanase Activity




       Andrew Pearson B. Sc. (Hons.), B. Beh. Sc.




                  Institute for Glycomics
         Griffith University (Gold Coast Campus)




Submitted in fulfilment of the requirements of the degree of
                   Doctor of Philosophy




                         July 2007
                                                                   ABSTRACT

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules located in the
extracellular matrices and basement membranes; they present a physical barrier to the
movement of cells (e.g., tumour cells and leukocytes) into tissues and play an important
role in a variety of biological processes including inflammation, the metastatic potential
of tumour cells, and angiogenesis. The basic HSPG structure consists of a protein core,
to which are attached several linear glycosaminoglycan (GAG) chains that confer most
of the biological properties of HSPGs.


Cleavage of heparan sulfate (HS) chains is critical for the modulation of the biological
function of HS-binding proteins, and profoundly affects cell and tissue function
involving migration and response to changes in the extracellular matrix. It is also
essential in the degradation of the extracellular matrix by invading cells, particularly
metastatic tumour cells and leukocytes entering inflammatory sites.


Heparanase is an endo- -D-glucuronidase that degrades HS-GAG chains; elevated
levels of heparanase expression correlates with metastatic potential, tumour vascularity,
and reduced post-operative survival of cancer patients. Heparanase inhibitors reduce the
incidence of tumour metastases; hence, heparanase is a promising target for anticancer
drug development. The most clinically advanced heparanase inhibitor, PI-88, is
currently undergoing clinical trials for melanoma, myeloma, and lung carcinoma.


The lack of a convenient, functional assay has hampered progress into the investigation
of heparanase. Although several heparanase assays have been developed, they rely on
either radiolabelled substrates or separation of enzymatically degraded substrates on the
basis of molecular size. The primary objective of this investigation was to develop a
simple fluorometric or colourimetric assay for heparanase activity. The development of
such an assay would be extremely beneficial in studies on heparanase, including the
kinetic evaluation of potential inhibitors and the correlation of heparanase with various
disease states.


Chapter One provides a general introduction to heparan sulfate, the synthesis of heparin
and heparan sulfate oligosaccharides, the structure and function of heparanase,


                                                                                         i
heparanase substrate specificity, inhibition of heparanase, current heparanase assays,
and the scope of the thesis.


A library of monosaccharide glucuronides and a library of disaccharide glycosides were
synthesised as putative heparanase substrates. These glycosides had various aryl
aglycons that could be measured spectrophotometrically upon hydrolysis of the
glycosidic linkage by an enzyme such as heparanase. The synthesis of these
monosaccharide and disaccharide libraries of aryl glycosides is detailed in Chapters
Two and Three, respectively.


In order to obtain sufficient quantities of heparanase for enzyme assays, human
heparanase cDNA was cloned into a baculovirus expression vector according to a
published procedure. The expression of heparanase in insect cells was contracted by the
industry partner, Progen Pharmaceuticals Ltd. Purification of recombinant human
heparanase was performed by Progen Pharmaceuticals Ltd. The cloning of human
heparanase is detailed in Chapter Four.


The evaluation of the monosaccharide and disaccharide libraries as heparanase
substrates is presented in Chapter Five. It was found that the N-sulfated 4-nitrophenyl
glycosyl glucuronide 154 and the N-sulfated methylumbelliferyl glycosyl glucuronide
158 were hydrolysed by recombinant human heparanase with specific activities of 17
and 48 nmol/h/mg, respectively. These compounds are the simplest substrates of
heparanase that have been reported, and may be beneficial in studies on heparanase,
including the kinetic evaluation of potential inhibitors and the correlation of heparanase
activity with various disease states.




Experimental data supporting the synthesis of the libraries of aryl glycosides presented
in Chapters Two and Three is detailed in Chapter Six. Selected 1H NMR spectra are
presented in the Appendix.




ii
           COMMUNICATIONS AND PUBLICATIONS

Communications
Pearson, A. G.; Kiefel, M. J.; Ferro, V.; von Itzstein, M. Towards the development of
probes for heparanase. 22nd International Carbohydrate Symposium 2004, Glasgow,
Scotland. Poster Presentation.


Pearson, A. G.; Kiefel, M. J.; Ferro, V.; von Itzstein, M. Towards the synthesis of aryl
glucuronides as heparanase probes. Brisbane Biological and Organic Chemistry
Symposium 2005, Brisbane, Australia. Oral Presentation.




Publications

Pearson, A. G.; Kiefel, M. J.; Ferro, V.; von Itzstein, M. Towards the synthesis of aryl
glucuronides as potential heparanase probes. An interesting outcome in the
glycosidation of glucuronic acid with 4-hydroxycinnamic acid. Carbohydrate Research
2005, 340 (13), 2077-2085.




                                                                                      iii
                              STATEMENT OF ORIGINALITY



This work has not previously been submitted for a degree or diploma in any university.
To the best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made in the thesis
itself. This thesis is less than 100,000 words in length, excluding appendices and
references.




Andrew Pearson
July 2007




iv
                                            TABLE OF CONTENTS

Abstract                                                                 i

Communications and Publications                                        iii

Statement of Originality                                               iv

Table of Contents                                                       v

Abbreviations                                                           x

List of Figures                                                       xvi

List of Tables                                                        xxi

Nomenclature                                                         xxii

Acknowledgements                                                     xxiii



                                                        CHAPTER ONE
  INTRODUCTION                                                          1

     Proteoglycans                                                      2

       Heparan Sulfate Proteoglycans                                    2

     Glycosaminoglycans                                                 3

       Heparan Sulfate Glycosaminoglycans                               4

       Heparin                                                          5

       Structure of Heparan Sulfate and Heparin Glycosaminoglycans      6

       Sequence Domains of Heparan Sulfate                              8

       Biosynthesis of Heparan Sulfate                                  9

     Glycosaminoglycan-Protein Interactions                            10

     Heparan Sulfate-Binding Proteins                                  11

       Heparan Sulfate-Degrading Enzymes                               11

     Heparanase                                                        13

       Heparanase structure                                            13


                                                                        v
        Molecular Modelling of Heparanase                          14

        Heparanase Biosynthesis and Trafficking                    16

        Heparanase Regulation and Normal Function                  17

        Role of Heparanase in Cancer Progression                   19

        Involvement of Heparanase in Tumour Metastasis             19

        Involvement of Heparanase in Tumour Angiogenesis           21

      Heparanase Substrate Specificity                             22

        Mechanism of Hydrolysis of Heparan Sulfate by Heparanase   24

      Inhibition of Heparanase                                     26

      Heparanase Assays                                            27

        Heparan Sulfate Labelling                                  27

        Size Difference-Based Methods of Assaying Heparanase       30

        Alternative Methods of Assaying Heparanase                 30

      Synthesis of Heparin and Heparan Sulfate Oligosaccharides    32

      Scope of Thesis                                              43



                                                         CHAPTER TWO
     SYNTHESIS OF A MONOSACCHARIDE LIBRARY OF PUTATIVE
     HEPARANASE SUBSTRATES
      Introduction                                                 45

        Anomeric control in glycosylation                          47

        Per-O-acetate couplings                                    48

        Glucuronosyl Bromide couplings                             48

        1-Hydroxy sugar couplings                                  49

        Trichloroacetimidate method                                50

        Other methods of glucuronide couplings                     50

        Oxidation of glucosides                                    51

        4-Methylumbelliferyl Glucuronide Synthesis                 51

vi
   Glucuronide Synthesis                                     52

 Results and Discussion                                      53

   Synthesis of Glucuronosyl Bromide 28                      54

   Glycosidation with Glucuronosyl Bromide 28                54

   Glucuronosyl Trichloroacetimidate 37                      55

   Synthesis of Non-Tagged Glucuronides                      57

   Synthesis of Tagged Glucuronides                          59

   Attempted Synthesis of Cinnamyl Glucuronide 62            61

   Proposed Mechanism for the Formation of 64 and 65         63

   Glucuronosyl Fluoride 66                                  65

   Synthesis of Cinnamyl Glucuronide 78                      67

   Deprotection of Glucuronides                              68

 Conclusion                                                  71



                                                  CHAPTER THREE
SYNTHESIS OF A DISACCHARIDE LIBRARY OF PUTATIVE
HEPARANASE SUBSTRATES
 Introduction                                                73

   Synthetic Strategy Towards Glycosyl Glucuronides          73

   Formation of the -Glucosamine Linkage                     75

   Glucosamine Donor 85                                      75

   Glucuronate Acceptor 86                                   76

 Results and Discussion                                      77

   Synthesis of the Glucosamine Trichloroacetimidate 85      77

   Synthesis of the Glucuronate Acceptor 86                  79

   Synthesis of GlcN- -(1→4)-GlcA Disaccharide               87

   Synthesis of Protected Glycosyl Glucuronides              96

   Deprotection, N-Acetylation, and N- and O-Sulfation       99

                                                             vii
       Conclusion                                                     109



                                                       CHAPTER FOUR
  CLONING OF HUMAN HEPARANASE
       Introduction                                                   111

       Experimental                                                   112

       Methods                                                        115

         Cloning of Heparanase Subunits                               115

         Insect Cell Expression                                       116

         Purification and Detection of Human Recombinant Heparanase   116

       Results                                                        117

       Discussion                                                     125



                                                        CHAPTER FIVE
  BIOLOGICAL EVALUATION
       Introduction                                                   127

          -Glucuronidase                                              128

       Methods                                                        129

          -Glucuronidase Assays                                       129

         Heparanase Assays                                            130

       Results and Discussion                                         132

          -Glucuronidase Assays                                       132

         Heparanase Assays                                            139




viii
 Conclusions                      145

 Future Directions                147



                         CHAPTER SIX
EXPERIMENTAL                      149




REFERENCES                        211




                           APPENDIX
            1
SELECTED H NMR SPECTRA            250




                                   ix
                                     ABBREVIATIONS

°C        degrees Celsius
2-NAP     2-napthylmethyl
A         adenine
Å         ångström
Ac        acetate
AGRF      Australian Genome Research Facility
Akt       protein kinase B
Ala       alanine
All       allyl
Alloc     allyloxycarbonate
ANU       Australian National University
aq.       aqueous
ATIII     antithrombin III
AU        absorbance units
B         biotin
BamHI     Bacillus amyloliquefaciens H (G GATCC)
BFA       bafilomycin A
BglII     Bacillus globigii (A GATCT)
BM        basement membrane
Bn        benzyl
bp        base pairs
BSA       bovine serum albumin
Bu        butyl
Bz        benzoate
C         cytosine
CA        California
Cbz       carboxybenzyl
CDB-FGF   cell-binding domain of human fibroblast growth factor
cDNA      complementary deoxyribonucleic acid
cHRG      chicken histidine-rich glycoprotein
CIP       calf intestinal alkaline phosphatase


x
conc.    concentrated
COSY     correlation spectroscopy
CS       chondroitin sulfate
CSA      camphorsulfonic acid
Cyto.D   cytochalasin D
d        day
DAST     diethylaminosulfur trifluoride
DBU      1,8-diazobicyclo[5.4.0]undec-7-ene
DEAD     diethyl azodicarboxylate
DMAP     4-dimethylaminopyridine
DMF      N,N-dimethylformamide
DMSO     dimethylsulfoxide
DNA      deoxyribonucleic acid
DTBMP    2,6-di-tert-butyl-4-methylpyridine
ECM      extracellular matrix
EcoRI    Escherichia coli RY13 (G AATTC)
ER       endoplasmic reticulum
ESI      electrospray ionisation
ESIMS    electrospray ionisation mass spectrometry
Et       ethyl
Eu       europium
FGF      fibroblast growth factor
FRET     fluorescence resonance energy transfer
FU       fluorescence units
g        gram
g        gravity
G        guanine
GAG      glycosaminoglycan
Gal      galactose
GH-A     glycoside hydrolase clan A
GlcA     D-glucuronic    acid
GlcN     D-glucosamine

GlcNAc   D-N-acetylglucosamine

Gln      glutamine
Glu      glutamic acid


                                                     xi
GPI       glycosylphosphatidylinositol
h         hour
HexA      hexuronic acid
HindIII   Haemophilus influenza (A AGCTT)
HMPT      hexamethylphosphoramide
HPLC      high performance liquid chromatography
HRMS      high resolution mass spectrometry
HS        heparan sulfate
HS-GAG    heparan sulfate glycosaminoglycan
HSPG      heparan sulfate proteoglycan
HSQC      heteronuclear multiple quantum coherence
HTRF      homogeneous time-resolved fluorescence
Hz        Hertz
IC50      half maximal inhibitory concentration
IdoA      L-iduronic   acid
Ile       isoleucine
J         coupling constant
kb        kilobase
kDa       kiloDalton
L         litre
LB        Luria-Bertani
Lev       levulinate
LG        leaving group
lit.      literature
LMW       low-molecular-weight
LRMS      low resolution mass spectrometry
Ltd.      limited
Lys       lysine
M         molar
MA        Massachusetts
MCA       monochloroacetate
mCPBA     meta-chloroperbenzoic acid
Me        methyl
MeOH      methanol
Met       methionine


xii
mg     milligram
MHz    megahertz
min    minute
mL     millilitre
mm     millimetre
mM     millimolar
mmol   millimole
mp     melting point
mRNA   messenger ribonucleic acid
MS     mass spectrometry
n      normal
N      normal
NA     N-acetylated
NA/S   N-acetylated/N-sulfated
NdeI   Neisseria denitrificans (CA TATG)
NJ     New Jersey
nm     nanometre
nmol   nanomole
NMR    nuclear magnetic resonance
NS     N-sulfated
NSW    New South Wales
PAPS   3'-phosphoadenosine-5'-phosphosulfate
PCR    polymerase chain reaction
Pfu    Pyrococcus furiosus
PG     protecting group
Ph     phenyl
Piv    pivaloate
PKC    protein kinase C
pMB    para-methoxybenzoate
POD    peroxidase
ppm    parts per million
Pr     propyl
Py     pyridine
QLD    Queensland
Rf     retention factor


                                               xiii
rpm       revolutions per minute
s         second
SA        streptavidin
sat.      saturated
SD        standard deviation
SmaI      Spore Membrane Assembly (CCC GGG)
SMARTTM   switch mechanism at the 5’ end of RNA transcript
sp        signal peptide
T         thymine
t1/2      half life
TBAF      tetra-n-butylammonium fluoride
TBDMS     tert-butyldimethylsilyl
TBDPS     tert-butyldiphenylsilyl
TCA       trichloroacetamide
TEMPO     2,2,6,6-tetramethylpiperidine-1-oxyl
tert      tertiary
Tf        trifluoromethanesulfonate
TFA       trifluoroacetic acid
THF       tetrahydrofuran
TIM       triosephosphateisomerase
TIPS      triisopropylsilyl
TLC       thin layer chromatography
TMS       trimetylsilyl
TMSEM     2-trimethylsilylethoxymethyl
Tol       tolyl
UK        United Kingdom
USA       United States of America
VEGF      vascular endothelial growth factor
VIC       Victoria
XhoI      Xanthomonas holcicola (C TCGAG)
Xyl       xylose
          alpha
          beta
          chemical shift in parts per million (NMR)
          epsilon

xiv
 g     microgram
μL     microlitre
 M     micromolar
μmol   micromole




                    xv
                                                        LIST OF FIGURES

Figure 1. The ECM is comprised of a network of proteins and polysaccharides.          1

Figure 2. Classes of cell surface HSPGs.                                              3

Figure 3. Members of the GAG family.                                                  4

Figure 4. Cell-surface HSPG.                                                          5

Figure 5. Major and variable disaccharide repeating units in HS and heparin.          6

Figure 6. Predominant conformations of residues in HS and heparin.                    7

Figure 7. Scheme of HS GAG biosynthesis.                                             10

Figure 8. Antithrombin-binding HS octasaccharide.                                    11

Figure 9. Cleavage of HS by the prokaryotic heparin lyases.                          12

Figure 10. Hydrolysis of HS by the eukaryotic heparanase.                            12

Figure 11. Structure and processing of human heparanase.                             14

Figure 12. Predicted model of the active heparanase heterodimer.                     15

Figure 13. Modelled heparanase with crystal xylanase and glycanase.                  16

Figure 14. Heparanase biosynthesis and trafficking.                                  17

Figure 15. Release of bioactive molecules as a result of heparanase hydrolysis of HS. 18

Figure 16. Metastasis.                                                               20

Figure 17. The role of heparanase in BM degradation by metastatic tumour cells.      21

Figure 18. Tumour angiogenesis.                                                      22

Figure 19. A minimally O-sulfated HS sequence required for substrate recognition by
heparanase.                                                                          23

Figure 20. Substrate specificity of human heparanase.                                24

Figure 21. Mechanisms of inverting and retaining glycosidases.                       25

Figure 22. Heparanase assay using biotinylated [3H]-HS immobilised on streptavidin
plates.                                                                              28

Figure 23. Heparanase assay using homogeneous time-resolved fluorescence.            29


xvi
Figure 24. HS degrading enzyme assay from Takara.                                      30

Figure 25. Hydrolysis of fondaparinux (3) by heparanase.                               31

Figure 26. Common features of glycosylation reactions.                                 33

Figure 27. Synthesis of the regular sequence disaccharides of heparin.                 35

Figure 28. Synthesis of heparin/HS disaccharides with "reversed" sequences.            35

Figure 29. Modular approach towards the assembly of heparin oligosaccharides.          36

Figure 30. Modular synthesis of heparin/HS oligosaccharides.                           36

Figure 31. Synthesis of -linked disaccharides.                                         37

Figure 32. Modular approach towards heparin oligosaccharides.                          37

Figure 33. Synthesis of a hexasaccharide using a [3 + 3] approach.                     38

Figure 34. Attempted [2 + 4 + 2] synthesis of an octasaccharide.                       39

Figure 35. Synthetic strategy for a heparin pentasaccharide.                           40

Figure 36. Synthesis of heparin/HS di- and tri-saccharides containing GlcA.            40

Figure 37. Orthogonal sulfation strategy for synthetic HS ligands.                     41

Figure 38. Disaccharide building blocks for the assembly of a heparin/HS library.      41

Figure 39. Chemoenzymatic synthesis of ATIII-binding HS pentasaccharide.               42

Figure 40. Minimum trisaccharide structure of the human heparanase cleavage site in
HS/heparin.                                                                            43

Figure 41. Hydrolysis of the tag generates a measurable response.                      44

Figure 42. Heparanase cleavage of the chromogenic or fluorogenic tag from 17 or 18
generates a measurable response.                                                       45

Figure 43. Preference for the 1-halide derivatives to be in the axial position.        47

Figure 44. Preparation of 1,2-trans glycosides by neighbouring group participation.    47

Figure 45. 4-Methylumbelliferyl glucuronide synthesis.                                 52

Figure 46. Retrosynthetic analysis of GlcA -linked to fluorogenic and chromogenic
moieties.                                                                              53

Figure 47. Partial 1H NMR spectrum of 55 in CDCl3 and CD3CN.                           58

Figure 48. Proposed mechanism for the formation of 64 and 65.                          63


                                                                                      xvii
Figure 49. Reported competitive reactivity of phenols versus carboxylic acids.        64

Figure 50. Reported synthesis of mannosyl fluoride 74.                                65

Figure 51. Possible rationale for the formation of the -fluoride 66.                  66

Figure 52. Retrosynthetic analysis of compounds of the general structure 17.          74

Figure 53. Synthesis of disaccharide 90 resulted in a mixture of anomers.             75

Figure 54. Glycosylation of 85 with 86 led exclusively to -linked disaccharide 91.    75

Figure 55. Synthesis of the GlcN glycosyl donor 85 from D-glucosamine.                76

Figure 56. Alternative route for the synthesis of the GlcN donor 85.                  76

Figure 57. Synthesis of conformationally locked GlcA acceptor 86.                     77

Figure 58. Alternative route for the synthesis of GlcA acceptor 86.                   77

Figure 59. Alternative approach for the synthesis of 94 from 50.                      82

Figure 60. Proposed reaction mechanism for the treatment of 113 with base.            84

Figure 61. Synthesis of trichloroacetimidate donor 117 from 85 and 86.                88

Figure 62. Proposed synthetic route from 123 to the disaccharide targets.             92

Figure 63. Alternative scheme towards the synthesis of the disaccharide library of
putative heparanase substrates.                                                       93

Figure 64. Favoured synthetic scheme towards the synthesis of the disaccharide library
of putative heparanase substrates.                                                    94

Figure 65. Deprotection, N-acetylation/N-sulfation and O-sulfation conditions for
compounds 135 – 138 were determined on the model compound 141.                       100

Figure 66. The disaccharide library of putative heparanase substrates.               110

Figure 67. Vector and restriction map of pCR-Blunt.                                  113

Figure 68. Vector map of pAcGP67-A, highlighting the secretion signal sequence and
multiple cloning site.                                                               114

Figure 69. Vector map of pAcUW51.                                                    114

Figure 70. Cloning strategy for the expression of active recombinant heparanase.     117

Figure 71. Gel electrophoresis of heparanase cDNA.                                   118




xviii
Figure 72. Gel electrophoresis of pCR-Blunt fused to the heparanase cDNA, digested
with BamHI.                                                                       118

Figure 73. PCR and purification of 8 kDa and 50 kDa subunits.                     119

Figure 74. Gel electrophoresis of cDNA encoding heparanase 8 kDa and 50 kDa
subunits.                                                                         120

Figure 75. Gel electrophoresis of heparanase subunits in preparation for ligation into
pAcGP67-A.                                                                        121

Figure 76. Gel electrophoresis of heparanase subunits excised from pAcGP67-A by
digest with SmaI and EcoRI.                                                       121

Figure 77. Gel electrophoresis of PCR products.                                   122

Figure 78. Gel electrophoresis of heparanase subunits plus secretory sequence excised
from pCR-Blunt.                                                                   123

Figure 79. Gel electrophoresis of cDNA encoding the 8 kDa subunit fused to the
secretory sequence, excised from pCR-Blunt by digestion with BglII.               123

Figure 80. Gel electrophoresis of pAcUW51:8:SS digested with HindIII.             124

Figure 81. Gel electrophoresis of pAcUW51:8:50 digested with BglII and NdeI.      125

Figure 82. Libraries of putative heparanase substrates.                           127

Figure 83. -Glucuronidase catalyses the hydrolysis of -glucuronides and GAGs. 128

Figure 84. Plot of concentration vs fluorescence for 4-methylumbelliferone.       132

Figure 85. Hydrolysis of 4-methylumbelliferyl glucuronide 20 by -glucuronidase. 133

Figure 86. Lineweaver-Burk plot of the hydrolysis of 20 by -glucuronidase.        133

Figure 87. Plot of concentration vs absorbance for 4-nitrophenol at 405 nm.       134

Figure 88. Hydrolysis of 4-nitrophenyl glucuronide 21 by -glucuronidase.          134

Figure 89. Lineweaver-Burk plot of the hydrolysis of 21 by -glucuronidase.        135

Figure 90. Plot of concentration vs absorbance for 3-nitrophenol, 4-hydroxycinnamic
acid and 5-hydroxyisophthalic acid.                                               136

Figure 91. Absorption spectra of 3-nitrophenol and 4-hydroxycinnamic acid.        136

Figure 92. Competitive inhibition of -glucuronidase.                              138

Figure 93. Plot of concentration vs fluorescence for 4-methylumbelliferone.       139

                                                                                  xix
Figure 94. Specific activity of heparanase towards putative substrates.        141

Figure 95. HS tetrasaccharides previously reported to be the smallest substrates
hydrolysed by heparanase.                                                      142

Figure 96. Competitive inhibition of recombinant human heparanase.             144

Figure 97. Inhibition of human heparanase by putative heparanase substrates.   145




xx
                                                        LIST OF TABLES

Table 1. Attempted formation of -fluoride 66 from trichloroacetimidate 37.      67

Table 2. Summary of 1H NMR data of deprotected glucuronides.                    71

Table 3. Summary of 1H NMR data of compounds used toward the synthesis of 86.   87

Table 4. Summary of 1H NMR data of GlcN methyl glycosides.                      103

Table 5. Primers used in the cloning of heparanase subunits.                    113

Table 6. Optimal and measured wavelength of aglycons used in enzyme assays.     137

Table 7. Activity of heparanase towards putative substrates.                    140

Table 8. 1H and 13C NMR data for compound 95 in CDCl3.                          169




                                                                                xxi
                                                     NOMENCLATURE

For the purpose of this thesis, atoms pertaining to the carbohydrate are numbered 1
through to 6, as illustrated below. Atoms of the glycoside are, in general, numbered
from the glycosidic linkage and are indicated by a prime. Exceptions to this numbering
system include methylumbelliferyl glycosides, which are numbered from the lactone, as
illustrated below. The allyl portion of cinnamyl glycosides are numbered 1" to 3" as
illustrated below.




Atoms of the cinnamyl glycoside with two GlcA units are assigned in a similar manner,
except the numbering of the allyl portion is reversed and the second GlcA unit is
numbered 1"' to 6"' as illustrated below.




Atoms of the disaccharides are assigned as GlcN 1 through to GlcN 6, and GlcA 1 to
GlcA 6 as illustrated below.




xxii
                                          ACKNOWLEDGEMENTS

I wish to sincerely thank my supervisors, Professor Mark von Itzstein, Dr Milton Kiefel,
and Dr Vito Ferro for their guidance, support, and encouragement throughout the course
of this project. Mark, I admire and appreciate the work you have put in over many years
to get the Institute for Glycomics to where it is today, and I am grateful for the
opportunity to undertake my PhD within your group. Milton, I am forever indebted to
you for the many hours you have spent instructing me in the lab, sharing your
knowledge of chemistry, and proof-reading my thesis. Vito, your expertise on this
project has been invaluable and I am extremely appreciative of the thoroughness at
which you proof-read my thesis.


I am grateful to Joe Tiralongo for his assistance with the molecular biology, enzyme
assays, and for proof-reading these sections of my thesis. Thanks go to Edward
Hammond who taught me cloning, purified the heparanase, and assisted with the
heparanase assays.


I would also like to thank several members of the Institute for Glycomics for their
friendship and support – Robin Thomson, Darren Grice, Jamie Rich, Alex Szyczew, and
Tasneem Islam, who provided assistance with chemistry, Jenny Wilson and Thomas
Haselhorst for assistance with NMR, Jeff Dyason for technical support and buying the
newspaper, Greg Tredwell for help with -glucuronidase assays, Sarah McAtamney and
Andrea Maggioni for help with molecular biology, Fiona Crone, Sharon Ackerman, and
Sally Tullberg for all aspects of administration, and to Regan Hartnell, Paul Madge Pat
Collins, and Tessa Daal for keeping it real. Thank you also to the many staff and
students, past and present, who made my time at the institute a memorable experience.


My deepest thanks go to my family for their support, encouragement and love. To
Carolyn and Bonnie, thank-you for your love, patience and understanding.


I wish to thank Chris Parish and Craig Freeman, ANU, for the donation of heparanase
cDNA. This work would not have been possible without the financial assistance of an
Australian post-graduate award (industry) and Progen Pharmaceuticals Ltd.



                                                                                   xxiii

								
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