Chemical glycoproteomics for identification and discovery of glycoprotein alterations in human ...

170 Pages·2017·11.59 MB·English

by  David Spiciarich

UC Berkeley
UC Berkeley Electronic Theses and Dissertations
Title
Chemical glycoproteomics for identification and discovery of glycoprotein alterations in
human cancer
Permalink
https://escholarship.org/uc/item/0t47b9ws
Author
Spiciarich, David
Publication Date
2017

Peer reviewed|Thesis/dissertation
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University of California
Chemical glycoproteomics for identification and discovery of
glycoprotein alterations in human cancer

by David Spiciarich

A dissertation submitted in partial satisfaction of the requirements for the degree
Doctor of Philosophy
in
Chemistry
in the
Graduate Division
of the
University of California, Berkeley

Committee in charge:
Professor Carolyn R. Bertozzi, Co-Chair
Professor David E. Wemmer, Co-Chair
Professor Matthew B. Francis
Professor Amy E. Herr

Fall 2017
Chemical glycoproteomics for identification and discovery of
glycoprotein alterations in human cancer

© 2017
by David Spiciarich
Abstract

Chemical glycoproteomics for identification and discovery of
glycoprotein alterations in human cancer

by

David Spiciarich

Doctor of Philosophy in Chemistry

University of California, Berkeley

Professor Carolyn R. Bertozzi, Co-Chair
Professor David E. Wemmer, Co-Chair

Changes in glycosylation have long been appreciated to be part of the cancer
phenotype; sialylated glycans are found at elevated levels on many types of cancer and
have been implicated in disease progression. However, the specific glycoproteins that
contribute to cell surface sialylation are not well characterized, specifically in bona fide
human cancer. Metabolic and bioorthogonal labeling methods have previously enabled
enrichment and identification of sialoglycoproteins from cultured cells and model
organisms. The goal of this work was to develop technologies that can be used for
detecting changes in glycoproteins in clinical models of human cancer.

In Chapter 1 of this dissertation, I present an overview of the structures and functions of
glycans and their relationship to cancer progression. I also discuss applications of in vivo
bioorthogonal labeling in model organisms and how in humans, the significant regulatory
and ethical barriers associated with introducing chemically altered sugars into people
have hindered it. Finally, I review mass spectrometry-based proteomics and how it can
be applied to clinical glycoproteomics.

In Chapter 2, I demonstrate the first application of this bioorthogonal labeling in a
glycoproteomics platform applied to human tissues cultured ex vivo. Both normal and
cancerous prostate tissues were sliced and cultured in the presence of functionalized
derivatives of N-acetyl mannosamine, the sialic acid biosynthetic precursor. Chemical
biotinylation followed by enrichment and mass spectrometry led to the identification of
glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue.
This work therefore extends the use of bioorthogonal labeling strategies to problems of
human clinical relevance.

Secretome proteins play important roles in regulation of many physiological processes
and show utility as potential biomarkers and for noninvasive diagnostics and treatment
monitoring. In Chapter 3, I discuss a platform for identifying sialoglycoproteins that were
secreted in the conditioned media from bioorthogonally labeled human prostate tissue
- 1 -
Abstract

slice cultures. This platform could be used to identify disease biomarkers in a faithful
clinical model of human disease.

Mutations in granulocyte colony-stimulating factor 3 receptor (CSF3R), also known as G-
CSFR, occur in the majority of patients with chronic neutrophilic leukemia (CNL) and are
more rarely present in other kinds of leukemia. In Chapter 4, I discuss novel variants in
CSF3R at asparagine residue N610, one of which was germline. Interestingly, these
N610 substitutions are potently oncogenic and result in ligand-independent receptor
activation. They confer activation of the JAK-STAT signaling pathway and concurrent
sensitivity to JAK kinase inhibitors. The N610 residue is part of a consensus N-linked
glycosylation motif in the receptor. Detailed mass spectrometry analysis demonstrates
that this site is occupied by both complex and complex bisecting glycans. Further
analysis demonstrates that N610 is the primary site of sialylation of the receptor. This
study demonstrates that membrane-proximal N-linked glycosylation is critical for
maintaining the ligand dependence of the receptor. Furthermore, it expands the
repertoire of potently oncogenic mutations in CSF3R that are therapeutically targetable
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This dissertation is dedicated to my family and friends for all their support.
With great appreciation to my mom, sister, and Giorgio.

- i -
Chemical glycoproteomics for identification and discovery of
glycoprotein alterations in human cancer

Table of Contents
List of Figures ................................................................................................................... iv

List of Tables .................................................................................................................... vi

List of Abbreviations ........................................................................................................ vii

Acknowledgments ............................................................................................................ ix

Chapter 1. Glycoproteomics and unnatural sugars to study protein
glycosylation .................................................................................................................... 1

1.1 Introduction .......................................................................................................... 1

1.2 Glycosylation is part of the cancer phenotype .................................................... 4

1.3 Mass spectrometry-based glycoproteomics ....................................................... 6

1.4 Unnatural sugars for bioorthogonal enrichment of glycans ................................ 9

1.5 Conclusion ........................................................................................................ 12

1.6 References ....................................................................................................... 13

Chapter 2. Bioorthogonal labeling of human prostate cancer tissue slice cultures
for glycoproteomics ...................................................................................................... 22

2.1 Introduction ........................................................................................................ 22

2.2 Results .............................................................................................................. 24

2.3 Discussion ........................................................................................................ 34

2.4 Methods and Materials ..................................................................................... 37

2.5 Code ................................................................................................................. 40

2.6 References ....................................................................................................... 43

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Table of Contents

Chapter 3. Enrichment of sialoglycoproteins from human prostate tissue slice
culture conditioned media ............................................................................................ 49

3.1 Introduction ........................................................................................................ 49

3.2 Results .............................................................................................................. 52

3.3 Conclusion ........................................................................................................ 60

3.4 Tables ............................................................................................................... 61

3.5 Methods and Materials ..................................................................................... 63

3.6 References ....................................................................................................... 68

Chapter 4. A novel germline variant in CSF3R reveals a N-glycosylation site that is
important in receptor regulation ................................................................................. 73

4.1 Introduction ........................................................................................................ 73

4.2 Results .............................................................................................................. 74

4.3 Conclusion ........................................................................................................ 81

4.4 Tables ............................................................................................................... 85

4.5 Methods and Materials ..................................................................................... 88

4.6 References ....................................................................................................... 93

Appendices ................................................................................................................... 96

Appendix Table 1. Proteomic dataset of proteins found in normal prostate tissue
slice cultures (TSCs) ....................................................................................................... 97

Appendix Table 1. Proteomic dataset of proteins found in cancer prostate tissue
slice cultures (TSCs) ..................................................................................................... 117

Appendix Table 2. Proteomic dataset of proteins found in benign prostate hyperplasia
prostatic tissue slice cultures (TSCs) ............................................................................ 142

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List of Figures

Figure 1-1. Common monosaccharides found invertebrate glycans along with
standard graphical representation ........................................................... 2

Figure 1-2. Examples of some classes of N-linked glycans covalently attached to
protein at asparagine (Asn) residues by an N-glycosidic bond ................ 3

Figure 1-3. Examples of protein O-glycosylation
  ........................................................ 4

Figure 1-4. Extracellular types of tumor-associated glycans
  ...................................... 5

Figure 1-5. A schematic of a typical bottom-up or shotgun proteomic experiment ..... 8

Figure 1-6. Metabolic bioorthogonal engineering ..................................................... 10

Figure 1-7. Bioorthogonal reactions with azides and terminal alkynes ..................... 11

Figure 2-1. Workflow for preparation and bioorthogonal labeling of human prostate
tissues slice cultures (TSCs) .................................................................. 23

Figure 2-2. Structure of azide-functionalized unnatural sialic acid precursor
monosacchride (Ac ManNAz) used for metabolic labeling of
4
sialoglycoproteins .................................................................................. 24

Figure 2-3. Histology of prostate tissue slice cultures (TSCs) by hematoxylin and
eosin (H&E) staining ............................................................................... 25

Figure 2-4. Western blot analysis of human prostate TSCs lysate administered
azide- and alkyne- functionalized sugars and reacted with corresponding
biotin probe under copper-catalyzed azide-alkyne [3+2] cycloaddition
(CuAAC) conditions ............................................................................... 26

Figure 2-5. Labeling and imaging of cell surface sialoglycoproteins in human prostate
tissue slice cultures (TSCs) ................................................................... 27

Figure 2-6. Efficient biotinylation of sialoglycoproteins using Ac ManNAz
  ............... 28

4

Figure 2-7. Distribution of identified proteins after enrichment
  ................................. 29

Figure 2-8. Voltage-dependent anion-selective channel 1 (encoded by the gene
VDAC1) is overexpressed in human prostate cancer tissue .................. 31

Figure 2-9. Legumain is over-expressed and glycosylated in human prostate cancer
tissue ...................................................................................................... 34

Figure 2-10. Comparative analysis of differentially regulated glycoproteins between normal
and cancer TSCs ...................................................................................... 36
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List of Figures

Figure 3-1. Dynamic range of proteins present in serum .......................................... 50

Figure 3-2. Schematic for acquisition of conditioned media (CM) from prostate tissue
slice cultures (TSCs)
  .............................................................................. 51

Figure 3-3. Western blot analysis of conditioned media (CM) collected from PC-3
cells incubated with Ac ManNAc (-) or Ac ManNAz (+) and subsequently
4 4
reacted with biotin-functionalized reagents ............................................. 52

Figure 3-4. Time independence of alkyne-biotin labeling on CM collected from PC-3
cells ........................................................................................................ 54

Figure 3-5. Concentration dependence of biotin-alkyne reaction on CM derived from
human TSC CM ..................................................................................... 56

Figure 3-6. Labeling of CM harvested from human prostate TSCs varies by collection
time
  ........................................................................................................ 58

Figure 3-7. Labeled glycoproteins from conditioned media administered Ac ManNAz
4
or Ac ManNAc ......................................................................................... 59
4

Figure 3-8. Optimized enrichment of labeled glycoproteins from conditioned media
administered Ac ManNAz or Ac ManNAc .............................................. 59
4 4

Figure 4-1. A novel germline mutation in CSF3R
  ..................................................... 75

Figure 4-2. The CSF3R N610H mutation is transforming and activates the JAK-STAT
pathway in a ligand-independent manner .............................................. 76

Figure 4-3. Annotated MALDI-TOF MS spectra of permethylated N-glycans from
CSF3R WT .............................................................................................. 77

Figure 4-4. Identification of sialic acid glycosylation on N610. Schematic of labeling of
sialic acid glycoproteins with an azide-functionalized sugar ................... 79

Figure 4-5. General scheme for stable isotope labeling by amino acids in cell culture
(SILAC) analysis of cellular proteome dynamics ................................... 80

Figure 4-6. A somatic N610S mutation identified in a patient with CNL is transforming
and therapeutically targetable ................................................................. 83

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