Glycomics in Biotechnology

Glycomics in Biotechnology
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How the technology works.
Glycomics is a new concept proposed during the 20 century following proteomics and genomics. It is the study of glycomes, which includes studying all the glycan structures of a cell type or any organism (Google, 2013). Glycans as antigens are often dominant. Substituents of the anomeric generate an electrophilic intermediate by acting as the leaving group. When a hydroxyl group that is a nucleophile reacts with this species, it leads to the glycosidic linkage formation. In this process oligosaccharide are formed amongst a multitude of functional groups ( hydroxyl and amino) with similar reactivity to allow access to branched structures. The conjugate vaccines are subunit vaccines that contain carbohydrates antigens that are linked to an immunogenic carrier covalently. According to researchers, malaria parasite has a malarial toxin that is responsible for the pathogenesis. At the surface of the malaria parasite is a large amount of glycosylphosphatidylinositols (GPIs) that anchors to the on the cell surface in the proteins. A glycan that is formed from reacting a synthetic pseudohexasaccharide, 2-iminothiolane and a sulfhydryl conjugates with a keyhole limpet haemocyanin activated by maleimide as protein carrier. This conjugate is used in vaccines to immunize.
How it was applied to solve a particular problem.
Malaria has been and still is one of the most devastating diseases in the tropics. With well over 40% of the world’s population living with the risk of malaria contraction. The conjugate containing vaccines serve as anti-toxin, which provides protection against pathogenesis.
What are the strengths and weaknesses of the new technology relative to one other technology that is currently used to address the same problem?
The science of biosynthetic pathways of glycans is complex and unlike the genomes that were used before, glycans are very dynamic. Otherwise sugars, have complex structures too as they are highly branching at the binding site with proteins and becomes difficult to decipher. On the other hand, unlike genomics and proteomics, which use template, glycomics use non-templates Carolyn. R & Ram. S, 2009).
The major strength of the glycomics technology is the fact that glycans play very many roles in physiology of bacterial. As such, it can be used to formulate next generation drugs and vaccines, bioactive glycans. Some of the most important medical applications include expanding cytolytic cells when treating cancer; as well avoid rejection when transplanting stem cells.

References
Carolyn R. Bertozzi and Ram Sasisekharan..(2009) Chapter 48Glycomics. Retrieved from HYPERLINK "/books/NBK1965/"/books/NBK1965/
Gooogle.(2013). glycomics definition. Retrieved from /search?q=using+Glycomics+in+Biotechnology.&ie=utf- 8&oe=utf-8&aq=t&rls=org.mozilla:en-US:official&client=firefox-a#client=firefox- a&hl=en&rls=org.mozilla:en- US:official&q=glycomics+definition&revid=1407975061&sa=X&ei=oQVoUYuJEoSDt AbftoDYDw&ved=0CJEBENUCKAA&bav=on.2,or.r_cp.r_qf.&fp=e00e1b3ddd47a221 &biw=1016&bih=356
Körber-Stiftung. (2007). Peter Seeberger – Prizewinner 2007.retrieved from -stiftung.de/en/science/koerber-european-science-prize/previous-prizewinners/2007.html
Link: /url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&ved=0CDMQFjAA&url=http%3A%2F%2F-institut.de%2Fbozen2006%2Fproceedings%2FSeeberger%2FSeeberger.pdf&ei=ly5pUdn5FcaMtQayxYCIAw&usg=AFQjCNF6vn9GmCLPCfu9tVHyf4QdWwEmgA&bvm=bv.45175338,d.Yms
Chemical Glycomics –
From Carbohydrate Arrays to a
Malaria Vaccine
Peter H. Seeberger
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH)
Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland
E-Mail: Seeberger@org.chem.ethz.ch
Received: 13th November 2006 / Published: 5th November 2007
Abstract
Chemical glycomics uses synthetic chemistry to procure defined carbohydrate
molecules to study the glycans involved in many functions
in the living cell. Based on an automated synthesis platform, a host of
synthetic tools including carbohydrate microarrays has been developed.
These tools have been employed to dissect carbohydrate-copolymer
interactions. Basic research in the glycomics arena is beginning
to impact on drug discovery, especially the development of carbohydrate-
based vaccines. The development of vaccine candidates to protect
from malaria and leishmaniasis infections is discussed.
Introduction
Glycomics is defined in analogy to genomics as the entire set of glycans produced in a
single organism. The realization that carbohydrates carry out important functions beyond
energy storage and providing structural stability of the cell wall is not new, but the tools
required to advance glycobiology at a more rapid pace were largely missing. Better, faster,
more reliable and more sensitive sequencing techniques for most glycoconjugates have
been developed in recent years [1]. Better sequencing allows for the comparison of carbohydrate
structures between different cell populations and the identification of relevant
carbohydrates even when those are present in very small amounts in a particular system.
An improved molecular understanding of specific structures resulted in a dramatically
increased need for defined molecules in quantities that are sufficient for biological, bio-
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Bozen 2006, May 15th – 19th, 2006, Bozen, Italy
Beilstein-Institut
chemical and biophysical studies. Speedy access to defined carbohydrates in sufficient
quantities is needed to gain access to oligosaccharides for the creation of tools that are
commonplace in genomics and proteomics.
Figure 1. Interactions of the three main biopolymers. (Reprinted from [2] with
permission of the RSC.)
Synthetic carbohydrates are needed to study carbohydrate-carbohydrate, carbohydrate-protein,
and carbohydrate-nucleic acid interactions (see Fig. 1). Rapid advances in the field of
glycomics have been hindered by the complexity of the biomolecules involved. Oligosaccharides
are structurally more complex than nucleic acids and proteins due to their frequent
branching and linkage diversity. The difficulty in isolating, characterizing and synthesizing
complex oligosaccharides has been a significant challenge to progress in the field.
Automated Synthesis of Oligosaccharides
Unlike the other major classes of biopolymers carbohydrates are often characterized by
highly branched motifs. Each monosaccharide unit has multiple sites of attachment to the
next sugar moiety. Additionally, each glycosidic linkage connecting two sugar units can
take on one of two possible isomeric forms. There are over one thousand different trisaccharides
possible when the ten mammalian monosaccharides are combined. The synthesis
of oligosaccharides has been pursued for over 100 years and the key coupling, the glycosylation
reaction is one of the most thoroughly studied transformations in organic chemistry
[3]. The anomeric substituent acts as a leaving group thereby generating an electrophilic
intermediate. Reaction of this species with a nucleophile, typically a hydroxyl group, leads
to the formation of a glycosidic linkage. A host of anomeric leaving groups has been
utilized in the construction of oligosaccharides. In addition to the formation of the glyco-
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sidic linkage, the multitude of functional groups (amino and hydroxyl) of similar reactivity
on each monomer emphasize the need for effective differentiation to allow for access to
branched structures. A plethora of protective groups for the masking of amino and hydroxyl
groups has been introduced. While much progress has been made, some linkages still
remain difficult to install. Particularly the synthesis of large, branched oligosaccharides
presents difficulties.
Enzymatic techniques rely on the high specificity of glycosyl transferase mediated glycosylations
and are an alternative to traditional chemical synthesis [4]. Utilizing nucleotide
diphosphosugars (NDPs) as building blocks, glycosyl transferases assemble complex carbohydrates
in aqueous media. A major advantage of this method is the ability to prepare
sophisticated structures without the need for protecting group manipulations on either the
building blocks or the desired product. While certain carbohydrates can be prepared using a
particular transferase, the narrow scope of transferase-mediated glycosylations necessitates
the isolation and purification of multiple enzymes to synthesize diverse structures.
The desire to streamline the synthesis of carbohydrates and allow non-experts to prepare
carbohydrates on demand has led to the design and evaluation of efficient one-pot methods.
A solution-phase orthogonal method that relies on thioglycosides as glycosyl donors is the
OptiMer_ strategy of Wong [5]. Analysis of the reactivity profiles for over a hundred
different thioglycosides using a computer program allows for prediction of the optimal set
of donors required to generate a given polysaccharide. The reactions are performed manually
in solution with the oligosaccharide chain grown from the non-reducing to the reducing
end.
Solution-phase oligosaccharide synthesis remains a slow process due to the need for
iterative coupling and deprotection steps with purification at each step along the way. Solid
phase synthesis has proven extremely efficient for the assembly of peptides and oligonucleotides
as it does not require purification after each reaction step, utilizes excess reagent
to drive reactions to completion and lends itself to automation. To alleviate the need for
repetitive purification events required during solid-phase oligosaccharide assembly, solidphase
techniques have been developed [6]. Different approaches to solid-phase oligosaccharide
synthesis had explored many critical aspects including the choice of synthetic
strategy, differentially protected glycosylating agents, solid support materials, and linkers
to attach the first monosaccharide to the support matrix.
We reported on the first automated solid phase oligosaccharide synthesizer in 2001 [7].
Attachment of the anomeric position of the reducing end sugar to the solid support allows
for the step-wise incorporation of one mono- or disaccharide building block at a time. The
use of UV active protective groups facilitates real time monitoring of the success of
automated synthesis as is common for the synthesis of peptides and oligonucleotides.
Glycosyl phosphates [8] and glycosyl trichloroacetimidates [9] proved to be useful building
blocks for the automated assembly that also incorporated novel linkers [10] to connect the
first sugar to the solid support. Based on innovative solutions to the protecting group, the
building block, the linker and the analysis challenge, the first automated synthesizer was
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based on a re- engineered peptide synthesizer [7]. Utilizing this automated synthesizer, a
host of biologically important oligosaccharides was prepared to demonstrate the power of
the approach. The synthesis of a complex carbohydrate like the Lex-Ley nonasaccharide
antigen that is found on tumour cells was accomplished in less than one day when compared
to well over one year using the most sophisticated solution phase synthesis methods
[11].
Scheme 1. Automated solid-phase synthesis of a Ley -Lex nonasaccharide.
The currently available automated synthesizer has accelerated access to many carbohydrates
several hundred-fold, still some sequences remain difficult to make and not all
oligosaccharides can be assembled on solid support yet. The most time consuming step
for the procurement of pure carbohydrates, is the synthesis of sufficient quantities of
building blocks. Those building blocks will become commercially available in the near
future and will greatly facilitate synthetic efforts. Improvements will include new building
blocks and methodologies to access all possible linkages, accelerated protocols for the
deprotection of synthetic oligosaccharides and better robotic systems that will facilitate
access to multiple carbohydrates in parallel.
Screening Interactions Involving Carbohydrates
Glycoconjugates naturally decorate the surface of mammalian cells and are involved in
cell-cell communication. Carbohydrate-protein interactions are common and carbohydratecarbohydrate
interactions have been implicated. Carbohydrates also have been found to
specifically interact with bacterial RNA as is the basis for the mode of action for aminoglycoside
antibiotics.
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Carbohydrate-protein interactions
Recently, high-throughput screening methods to determine carbohydrate-protein interactions
have been introduced rapidly [12]. Now, access to pure oligosaccharides is the limiting
factor. Automated procurement of synthetic sugars has enabled the development of
carbohydrate arrays. Many applications exist as the arrays can be used to discover novel
carbohydrate protein interactions, define the epitopes recognized by disease-related or
vaccine induced antibodies. Carbohydrate antigens for vaccine development can be identified
using carbohydrate arrays. Initial carbohydrate microarrays in our laboratory focused
on a panel of mannose containing oligosaccharides (Fig. 2a) [13]. The molecules were
selected based on the glycans that decorate the viral surface envelope glycoproteins of
HIV. The arrays were composed of a series of closely related structural determinants of
(Man)9(GlcNAc)2. Using these arrays, precise profiles of the carbohydrate binding capacity
of a series of gp120 binding proteins (DC-SIGN, 2G12, Cyanovirin-N and Scytovirin) were
determined (Fig. 2b).
Figure 2a. Synthetic substructures of the triantennary N-linked mannoside including a
thiol-linker for immobilization. (Adopted from Chem. Biol. (2004) 11: 875 – 881)
[13].
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Figure 2b. Carbohydrate microarrays containing synthetic mannans 1 through 7 and
galactose, printed at 2 mM. False colour image of incubations with fluorescently
labelled ConA, 2G12, CVN, DC-SIGN and Scytovirin [13].
Figure 2c. Comparison of the binding profiles of fluorescently labelled Cyanovirin-N
and Scytovirin, with mannans 1 through 7 [13].
The binding profiles of multiple proteins can be established by presenting various structural
determinants of an important glycan on a single array, multiple proteins can be screened to
determine their binding profiles. Figure 2b illustrates the carbohydrate of two potent HIVinactivating
proteins isolated from cyanobacterium, Cyanovirin-N (CVN) and Scytovirin
[13]. The results illustrate that these two proteins recognize different structural motifs
within the high-mannose series of structures arrayed. A single experiment yields significant
data and saves much time compared with conventional methods.
Carbohydrate-RNA interactions
Aminoglycosides are carbohydrate antibiotics that contain amino sugars and are composed
of two to five monomers. Clinically, these compounds are used as broad-spectrum antibiotics
against a variety of important bacteria. The antibacterial effect is based on binding
of the aminoglycosides to the bacterial ribosomes, thereby inhibiting protein synthesis.
Therapeutic efficacy of aminoglycosides, however, has decreased recently due to increased
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antibiotic resistance. In recent years, the incidence of resistant bacteria has increased. In
order to combat the growing threat that bacteria pose to human safety, new antibiotics must
be identified [14].
Figure 3. Fluorescence intensity of slides incubated with 10 mM of human (18S) and
bacterial (16S) hairpin mimics of the A-site in rRNA.
Aminoglycoside microarrays were constructed by non-specific immobilization of the antibiotics
onto amine reactive glass slides using a DNA arraying robot [15]. This versatile
platform was used to probe the interactions of aminoglycosides with a variety of targets.
Arrays were probed with an RNA mimic of the bacterial and human A-sites (Fig. 3).
Different RNA sequences were used to establish the microarray method to screen for
RNA binding and binding specificity.
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Carbohydrate arrays to detect bacteria [16]
Carbohydrate arrays can help to determine the carbohydrate binding specificity of intact
bacterial cells. The carbohydrate-coated array surface presents carbohydrate ligands in a
manner that facilitates multivalent binding. Cell adhesion on the arrays can be readily
visualized using cell-permeable fluorescent dyes to stain the cells' nucleic acids. The
array-based method enables assay miniaturization and requires only minimal amounts of
ligand and cells when compared to solution measurements or experiments in 96-well plates.
Carbohydrate-cell interactions can be detected in homogeneous and heterogeneous solutions
that contain bacteria. Reliable detection even in complex mixtures that mimic body
fluids illustrates the potential this method may hold in the future as a pathogen-specific
diagnostic test.
Figure 4. An image of a carbohydrate array after incubation with ORN178 cells that
were stained with SYTO 83 cell-permeable nucleic acid staining dye. Each concentration
was spotted with three rows of five spots. Each spot is the result of delivery of
1 nL of a 20 mM, 5 mM, 1.25 mM, 310 mM, 63 mM, or 15 mM carbohydrate-containing
solution. The spot diameter is ~ 200 mm.
Synthetic Carbohydrate-Based Vaccines
Vaccines are the most powerful and cost-efficient medical intervention in the control,
prevention, and elimination of human infectious diseases [17]. Remarkable progress in
the development of vaccines against many different human pathogens including polio,
influenza, measles, diphtheria, tetanus, pertussis, varicella, mumps, rubella, hepatitis B,
Pneumococci, and Haemophilus influenza type B, has been made [18]. Vaccination has
enabled the eradication of smallpox and has decreased the incidence of once common
childhood diseases.
Despite these successes, bacterial and viral infections in humans still represent major health
problems, killing at least 15 million people annually. The search for new prophylactic and
therapeutic vaccines to combat these infections has attracted considerable attention [19],
albeit with only limited success. No effective vaccines against human parasites such as
malaria, leishmaniasis, and schistosomiasis exist [20]. Vaccines such as the Bacillus Calmette
Guerin (BCG) vaccine against tuberculosis, are often of limited efficacy. Thus, it is
crucial to improve our understanding of the relevant glycan and protein antigens.
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Vaccination is a way of inducing resistance to a foreign micro-organism by specially
training the immune system. The body is exposed to innocuous biological material that
mimics the infectious agent, but does not lead to infection or serious disease. The immune
system is stimulated to generate antigen-specific antibodies and to neutralize the antigens.
Subunit vaccines contain only parts of the micro-organism. Antigenic protein or carbohydrate
fragments are either purified from natural sources or produced synthetically. Subunit
vaccines against Haemophilus influenza type B,...