Download Targeted Identification of Glycoproteins in Disease

A targeted study of bacterial glycoproteins using metabolic oligosaccharide engineering
Emily Clark, 2015
Many species of pathogenic bacteria are developing resistance to antibiotic treatments.(1) As
such, there is a rising need for novel therapeutic targets. The glycosylated, or sugar-modified, proteins
present on bacterial cell surfaces provide one such target.(2) Importantly, glycosylation patterns vary
between bacterial and mammalian species, as well as among different bacterial species. (3) This variation
creates the potential to selectively target a bacterial species based on cell-surface glycans, while leaving
other cells unharmed.(4) Despite their potential as therapeutic targets, bacterial glycoproteins remain
largely unexplored. This lack of exploration is because, until recently, it was believed that bacteria did not
add sugars to their proteins.(5) Once this belief was refuted, though, bacterial glycoproteins proved
difficult to study with existing methods due to their unusual structures. Traditional methods used to detect
glycoproteins in human cells do not translate well to studying bacterial glycoproteins.(5) A method that
would ease the study of bacterial glycoproteins could help to vastly improve our knowledge of these
biomolecules and potentially reveal new targets of therapeutic intervention.
Previous work in the Dube lab has established metabolic oligosaccharide engineering (MOE) as
an exciting method for studying and targeting bacterial glycoproteins. MOE takes advantage of natural
biosynthetic pathways in bacteria and hijacks this machinery to metabolically incorporate an unnatural
sugar that contains a chemical handle into cellular glycoproteins. The incorporated chemical handle can
then be used for both visualization and potential targeting purposes (Figure 1).(6)
The Dube lab has shown incorporation of the unnatural sugar peracetylated Nazidoacetylglucosamine (Ac4GlcNAz) in the pathogenic bacterial species Helicobacter pylori and
Campylobacter jejuni.(4,7) The goal of my project was to use these methods to determine the localization
and identity of these glycoproteins. Further, I aimed to expand the use of MOE to explore the extent to
which a panel of unnatural sugars is incorporated into glycoproteins in a range of bacterial and
mammalian cells.
If glycoproteins are localized to the surface of a cell, it means they are vulnerable to targeting in a
therapeutic treatment. Ac4GlcNAz, for example, incorporates onto cell-surface proteins of H. pylori, but
not mammalian cells, creating the potential to selectively target H. pylori, while leaving host cells
unharmed.(4) Therefore, if a glycoprotein labeled with Ac4GlcNAz in C. jejuni was only located on the
interior of the cell, it means that this sugar could potentially be used to selectively target H. pylori and not
C. jejuni. However, my results suggest that Ac4GlcNAz is incorporated onto cell-surface proteins in C.
jejuni, meaning that Ac4GlcNAz cannot act as a selective therapeutic target for H. pylori. A different,
more selectively incorporated sugar would be therefore necessary for specific targeting.
Because of this, it is beneficial to examine the incorporation of other unnatural sugars across a
broad spectrum of both bacterial and mammalian species to possibly identify more selective sugars. To
aid in this process, the Dube lab received a shipment of unnatural sugars from Suvarn Kulkarni at the
Indian Institute of Technology including BacAz2, FucAz, and 6-Az-GlcNAc. My preliminary results
suggest that BacAz2 is incorporated onto the surface of C. jejuni, yet is absent from the surface of
mammalian cells. Additional results suggest that BacAz2 is incorporated in H. pylori. Further work will
examine the localization of this sugar in H. pylori, as well as other pathogenic bacteria, including
Burkholderia thailandensis and Bacteroides fragilis, which have previously been shown to not
incorporate Ac4GlcNAz.(4)
While Ac4GlcNAz may not serve as a selective therapeutic target, I worked to explore it’s utility
in studying the glycoproteins of bacteria which do incorporate it. Specifically, I worked towards
identifying the glycoproteins of C. jejuni that have been labeled with Ac4GlcNAz. I employed “Click-iT”
chemistry to isolate the labeled proteins. In essence, reactive beads were used to attract cells labeled with
the unnatural sugar using very selective chemistry. All non-labeled proteins were washed away. In the
future, I will analyze these samples via liquid chromatography/mass spectrometry to confirm C. jejuni
protein presence. The samples will then be sent to a collaborating lab to be analyzed via multidimensional
protein identification technology (mudPIT) to identify the glycoproteins. These proteins can then be
compared to previously established lists of C. jejuni glycoproteins, thereby assessing the utility of MOE
in glycoprotein identification. Ultimately, continuation of this two-fold project has the potential to expand
our knowledge of bacterial glycoproteins while identifying optimal metabolic substrates for antibiotic
treatment.
Figure 1. A schematic representing the process of metabolic oligosaccharide engineering (MOE) followed
by a Staudinger ligation to attach a phosphine probe to be used for a number of purposes, such as for
detection or immune stimulation. Adapted from figure by Kaewsapsak et al.(4)
Faculty Mentor: Danielle Dube
Funded by the Surdna Foundation Research Fellowship
References
1.
2.
3.
4.
Tomasz, A. (1994) Multiple-Antibiotic-Resistant Pathogenic Bacteria - A Report on the
Rockefeller University Workshop. N Engl J Med 330, 1247-1251
Dube, D. H., Champasa, K., Wang, B. (2011) Chemical tools to discover and target bacterial
glycoproteins. Chem. Commun., 87-101
Herget, S., Toukach, P.V., Ranzinger, R., Hull, W.E., Knirel, Y.A., von der Lieth, C. (2008)
Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): Characteristics
and diversity of bacterial carbohydrates in comparison with mammalian glycans. BMC Structural
Biology 8
Kaewsapsak, P., Esonu, O., Dube, D.H. (2013) Recruiting the host's immune system to target
Helicobacter pylori's surface glycans. ChemBioChem 14, 721-726
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Champasa, K., Longwell, S.A., Eldridge, A.M., Stemmler, E.A., Dube, D.H. (2013) Targeted
Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp).
Molecular and Cellular Proteomics 12, 2568-2586
Dube, D. H. (2012) Metabolic Labelling of Bacterial Glycans with Chemical Reporters. in
Bacterial Glycomics: Current Research, Technology and Applications (Reid, C. W. ed.), Horizon
Scientific Press. pp 229-242
Helble, J. (2014) Honors Project: An investigation of the efficacy of metabolic oligosaccharide
engineering in different bacterial species.