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3
Glycoscience in Health,
Energy, and Materials
Glycoscience contributes in fundamental ways to three key areas on
which the committee focused: the understanding of human health and
disease, the search for alternative sources of energy, and the development
of new materials. The committee selected these areas because they illus-
trate the range and diversity of research encompassed by glycoscience
as a field. They also help illustrate how glycoscience knowledge will be
embedded in efforts to address fundamental challenges in health and
sustainability.
The chapter begins with examples and questions related to human
health because this has been a major focus of efforts in the field of glyco-
science and glycomics, particularly in the United States. Indeed, many sci-
entists may automatically think of health when they think of glycans and
their functions. Although other researchers actively study carbohydrates
and their uses (e.g., in polymer engineering), the terminology and tech-
niques used by these fields may vary. As a result, the scientific commu-
nity may not immediately think of the totality of glycan research as part
of a unified field of glycoscience. One goal of this report is to provide a
view of glycoscience that encompasses a broader range of topics. Indeed,
although health care remains an important driver for research, increased
attention is being paid to other drivers, including the environment and
energy security (Johnson 2012), and glycoscience will be relevant in mul-
tiple contexts.
37
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38 TRANSFORMING GLYCOSCIENCE
3.1 GLYCOSCIENCE AND HEALTH
Over the past several decades, research from many laboratories has
established that glycans are directly involved in normal physiology and in
the etiology of every major disease afflicting mankind (Varki et al. 2009).
Deciphering the glycome creates an expanding frontier for knowledge
and discovery about human health. The section begins with an explana-
tion of the roles of glycans in fundamental biological processes, such as
inflammation and immune system activation, and moves on to consider
examples from infectious diseases and vaccine development. It then turns
to chronic diseases such as diabetes and cardiovascular disease and to a
discussion of cancer and congenital genetic disorders. Finally, the signifi-
cance of glycans in the development of new pharmaceuticals is discussed.
Examples of the diverse roles that glycans play in human health are
provided to illustrate the breadth and importance of glycoscience to this
field. The section does not attempt to comprehensively address all glycan
functions. As it illustrates, however, the development of a more complete
understanding of glycans can impact the diagnosis and treatment of infec-
tious, chronic, and genetic diseases. (See Figure 3-1 for a partial summary
of some of the roles played by glycans in biological systems.)
3.1.1 Glycans' Regulation of Inflammation
Inflammation, both chronic and acute, underlies the pathology of
a broad range of diseases, including diabetes, cancer, arthritis, asthma,
heart disease, and infectious disease (Barreiro and Sanchez-Madrid 2009;
Celie et al. 2009; Kobayashi et al. 2009a; Korpos et al. 2009; Langer and
Chavakis 2009; Schauer 2009; Sperandio et al. 2009; McEver 2010; McEver
and Zhu 2010; Sorokin 2010; Zarbock et al. 2011). Glycans play a key role
in inflammation at many levels.
Inflammation begins with the generation of multiple cytokines by
various cell types that react to pathogen-associated molecular patterns
(PAMPs) or danger-associated molecular patterns (DAMPs) arising
from damaged tissues. Many of these molecular patterns are glycocon-
jugates, and many cytokines themselves bind to endogenous glycans.
More recently recognized is the fact that glycans found in an individual
host, such as a human, can serve as self-associated molecular patterns
(SAMPs) that dampen inflammation and that SAMPs can be mimicked
by microbes (mSAMPs; Varki 2011). The signaling associated with these
molecular patterns is part of the multistep process that results in leu-
kocyte homing into affected tissues, a process initiated when leuko-
cytes adhere to activated endothelial cells lining blood vessel walls. This
Velcro-like adhesion slows the rapidly flowing leukocytes, causing them
to roll along the surfaces of the endothelial cells. Rolling leukocytes are
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 39
FIGURE 3-1 Glycans play diverse roles within biological systems.
SOURCE: Reprinted from Hart and Copeland 2010, with permission from Elsevier.
Figure 3-1
able to bind tightly to glycoprotein receptors, called integrins, which can
Bitmapped
lead to penetration of the endothelial cell monolayer and its basement
membrane. The mechanisms for the initial binding and rolling of leuko-
cytes have been studied extensively and involve transient expression of
highly regulated glycan-binding proteins, called selectins, that decorate
the surfaces of leukocytes, activated platelets, and activated endothelial
cells. Selectins are exquisitely specific at binding to certain glycan struc-
tures. One of the primary determinants for their binding specificity is
the tetrasaccharide known as sialyl Lewis x. The unique properties of
the interactions between a selectin protein and its specific glycan ligand
are critical to slowing leukocytes down so that they can bind to and then
extravasate into inflamed tissue. Selectins and their glycan ligands also
play a role in tumor metastasis (Dube and Bertozzi 2005; Laubli and
Borsig 2010; St Hill 2011).
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40 TRANSFORMING GLYCOSCIENCE
Glycoconjugates such as collagens, laminins, and sulfated proteo-
glycans that surround endothelial and other cells also play critical roles
in immune cell infiltration of tissues. Leukocytes secrete hydrolases,
enzymes that degrade extracellular matrix glycoconjugates and release
bioactive glycan-containing fragments in the extracellular milieu. These
glycan-containing fragments help perpetuate inflammation by affecting
leukocyte chemotaxis, activation, and differentiation. For example, frag-
ments of the polysaccharide hyaluronan are pro-inflammatory as a result
of their ability to bind to a class of receptors called Toll-like receptors and
serve as "danger signals" of acute cell injury or infection.
The steps in inflammation that are controlled by glycans and glyco-
conjugates represent potential novel targets for therapeutics that could
improve on current treatments like broad-acting steroids. In addition,
advances in medicinal chemistry now allow for the rational design of a
class of drugs--glycomimetics--that are based on the bioactive confor-
mations of functional glycans (Imberty et al. 2008; Ernst and Magnani
2009; Magnani and Ernst 2009; Garber et al. 2010; Chabre et al. 2011;
Drozdova et al. 2011; Jandus et al. 2011). For example, cell adhesion medi-
ated by selectins underlies the vascular occlusion crises that characterize
sickle cell anemia. One company has recently designed a small-molecule
antagonist that binds to selectins and is now in clinical trials as a treat-
ment for this disease. Other companies are investigating the use of anti-
bodies directed against a selectin and its ligand. Meanwhile, the already
approved drug heparin is known to block inflammation by blocking
selectin interactions. As a result of these advances, the study of functional
glycans represents a source of leads for novel therapeutics for treating
inflammation and a variety of other human diseases. The key roles played
by glycans in inflammation and the trafficking of white blood cells to
tissues also helped stimulate interest in understanding the physiological
importance of glycans in cell adhesion and cell signaling more broadly.
3.1.2 Glycans' Essential Role in Regulation of the Immune System
Inflammation is one result of the immune system's response to dan-
ger. Immunity is the other. Antibodies themselves are glycosylated pro-
teins, and gycans can also be the targets (antigens) for antibody binding
and the generation of immune responses.
The general importance of glycans in immunity has been appreciated
for many years, and the early discovery that the ABO blood groups derive
from specific glycan structures is just one example (Morgan and Watkins
1969). While in most cases the glycans that form part of antibodies do
not play a direct role in their binding to antigens, they do play a critical
role in their effector functions to activate components of the immune
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 41
system (Raju 2008; Lux and Nimmerjahn 2011). Mammals have devel-
oped sophisticated glycan recognition systems to recognize pathogen-
associated molecular patterns, including specific types of glycan-binding
proteins such as lectins, and Toll-like receptors. In fact, the most common
phenotype in mice that survive the selective deletion of glycosyltrans-
ferases is defective immune cell function (Marth and Grewal 2008). In
humans, dendritic cells play a primary role in the presentation of for-
eign antigens to the immune system (Erbacher et al. 2009). The glycans
on dendritic cell surfaces and multiple lectins are involved in dendritic
cell functions, including antigen uptake, immune modulation, detection,
processing of viral antigens, and trafficking (Silva et al. 2012). Meanwhile,
host glycans such as sialic acids serve as self-associated molecular pat-
terns. It is becoming evident that many of these glycan-protein interac-
tions are involved in the critical balance between immune tolerance and
the generation of a strong immune response.
Some human autoimmune diseases involve auto-antibodies that rec-
ognize self-glycans. The glycans on a pathogen may be sufficiently similar
to a human glycan such that an immune response to the pathogen leads
to the generation of cross-reactive antibodies. For example, this is thought
to be the case in the development of Guillain-Barré syndrome and Miller
Fisher syndrome (Kaida et al. 2009). The significance of antiglycan anti-
bodies is also being explored in other autoimmune diseases, such as sys-
temic lupus erythematosus and rheumatoid arthritis (Dai and Gao 2011;
Fattal et al. 2010; Louthrenoo et al. 2010).
The specific branching pattern of N-glycans on T-cell antigen recep-
tors regulates their threshold of activation, and a deficiency of a key
N-glycan branching glycosyltransferase known as GnT5 may contribute
to autoimmune disease (Lee et al. 2007). During their biosynthesis, B-
and T-cell antigen receptors are glycosylated in a manner reflective of
the cell's physiological state. This glycan plasticity alters their molecular
interactions at the cell surface, associations with signaling complexes,
and internalization via endocytosis. These glycans also appear to control
the spatial organization of receptors laterally in the plasma membrane.
Galectins, which are multivalent glycan-binding proteins, and their gly-
can ligands have numerous roles in varied immune processes, including
pathogen recognition, regulation of inflammation, and modulation of the
adaptive immune response (Rabinovich and Toscano 2009). There are
currently more than 15 different galectins known in the human genome,
each with varied glycan-binding specificity and distinct cellular distribu-
tions. Specialized galectins also modulate the threshold of T-cell receptor
activation during T-cell development (Demotte et al. 2008). In addition
to their role in regulating immune functions, galectins mediate cellular
interactions with parasites, viruses, bacteria, and fungi. Recent studies
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42 TRANSFORMING GLYCOSCIENCE
have indicated that glycan-galectin lattices on the surfaces of immune
cells modulate receptor signaling and play a role in modulating effector
functions (Rabinovich et al. 2007).
Another class of glycan-binding proteins--siglecs--also have critical
functions in immunity. Siglecs are membrane-bound, sialic-acid-binding,
immunoglobulin-like, glycan-binding proteins that play a key role in
regulating immune cell adhesion, signaling, and endocytosis (Crocker
et al. 2007; Crocker and Redelinghuys 2008). Sialic acids are negatively
charged monosaccharides that often appear at the terminus of glycan
structures. Siglec interactions with immune cell receptors at the cell sur-
face help inhibit abnormal immune cell activation. Siglecs are important
for preventing autoimmunity, and they influence the responses of almost
every cell in the immune system. There are currently 17 known siglecs
encoded in primate genomes, each with a different immune cell type
distribution and function. The types of sialyl-oligosaccharides and the
structures of the sialic acids on the surfaces of immune cells also play key
roles in the activity of siglec regulation of immune cell functions. Given
the very recent finding of sialic acids as self-associated molecular patterns
that help regulate these immune reactions, it is reasonable to suggest that
other self-glycan patterns might yet be discovered.
For high-affinity binding, siglecs require the glycans to be clustered
in specific patterns that are not well understood. Critical to their func-
tions is the ability to bind sialic acid glycans both on the same cell ( cis)
and on a different cell or microorganism (trans). The molecular mimicry
of host sialo-glycans by a variety of pathogens and beneficial microor-
ganisms takes advantage of siglecs. As is the case for the selectins, siglec-
specific agonists and antagonists represent potentially powerful, but as
yet untapped, targets for the development of therapeutics to treat auto-
immune and inflammatory diseases. Meanwhile, siglecs already serve
as targets for delivering chemotherapeutic agents to specific cell types.
For example, CD33, also known as Siglec-3, is the target of an antibody
approved for treatment of acute myeloid leukemia, and antibodies tar-
geting CD22/Siglec-2 are in clinical trials for treatment of B-cell non-
Hodgkin's lymphoma and autoimmune diseases (O'Reilly and Paulson
2009).
Glycans in the nucleus and cytoplasm also play a critical role in the
regulation of immunity. Recent studies have shown that O-GlcNAcylation,
a ubiquitous monosaccharide modification of nuclear and cytoplasmic
proteins (Hart et al. 2011), plays a key role in both T- and B-lymphocyte
activation (Golks et al. 2007). O-GlcNAc transferase is required for early
activation of B-lymphocytes via the B-cell receptor. Data suggest that
O-GlcNAcylation is required for nuclear translocation and functions of key
transcription factors regulating B-lymphocyte activation and functions.
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 43
Although there is still much to be learned about the roles of glycans
in immunity, new insights could yield important advances in treating a
wide variety of human diseases (Kolarich et al. 2012).
3.1.3 Glycans' Key Role in Infectious
Diseases and Vaccine Development
While glycans are important in regulating immunity, they are also key
actors in the constant battle between our cells and invading pathogens,
including viruses, bacteria, and parasites. Indeed, glycans are the domi-
nant molecules at this interface. Glycans and glycan-binding proteins, in
part because of their plasticity and rapid evolution, play a critical role on
both sides of this battle in nearly every species of pathogen (Bardoel and
van Strijp 2011). Not only are glycans commonly used by microbes and
viruses to bind to and infect host cells but also nearly all of the vaccines
for infectious diseases recognize glycans present on the disease-causing
organism. The complex mucin-bound glycans lining the epithelial sur-
faces of the human body not only block invasion by pathogens but also
provide binding sites essential to colonization by beneficial bacteria that
reside in our bodies and are required for our survival.
Many, if not most, bacteria have adhesins on their surfaces that bind
to cells via glycans. These protein-glycan interactions often determine the
tissue selectivity of bacterial pathogens (Pieters 2011). For example, lung
and airway pathogens, such as Pseudomonas aeruginosa, Haemophilus influ-
enza, and Staphylococcus aureus, primarily recognize glycans terminating
in GalNAc1-4Gal structures. Recent studies of the bacteria responsible
for gastric ulcers (Helicobacter pylori) indicate that these bacteria bind
sialylated glycans, such as those found on mucins and gangliosides in
the stomach (Kobayashi et al. 2009b). Conversely, the innate immune
system of humans, which is a major line of defense against pathogens,
has evolved to deal with millions of species of bacteria, fungi, and viruses
primarily by recognizing their foreign glycoconjugate structures (Bardoel
and van Strijp 2011). For example, the lipid A component of bacterial
lipopolysaccharide and the complex mannan structures on fungi are par-
ticularly potent elicitors of an immune response. The C-type lectins made
by our cells are an important component of our innate immunity in that
they recognize a wide variety of glycans on pathogens.
Research has confirmed only recently that the complex glycans pres-
ent in human milk play a role in protecting newborns from infections
and represent a major form of innate immunity (Newburg et al. 2005).
The variety of different glycan structures in human milk is enormous,
and recently developed glycomic methods are beginning to elucidate
the human milk glycome (Chichlowski et al. 2011; Tao et al. 2011). Milk
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44 TRANSFORMING GLYCOSCIENCE
glycans serve as soluble receptors for pathogens, in much the same way
that glycans on epithelial mucins function to inhibit pathogens from bind-
ing to the mucosal surface of the gastrointestinal tract. These and other
findings suggest that more detailed knowledge about milk glycans might
lead to novel antimicrobial agents to prevent, rather than treat, infections.
Because all human cells are covered with a thick glycocalyx, nearly all
pathogens, including viruses, must gain entry to their target cells by inter-
acting with glycans. The importance of glycans in influenza infection has
been known since the 1940s (Karlsson 1998). In recent years, however, the
critical roles of specific glycans in viral infections have been highlighted
by fears of a new influenza pandemic. The first step in flu virus infection
is the binding of a viral coat glycoprotein--hemagglutinin (HA)--to gly-
can structures on the host cell. Small mutations in HA enable it to bind
to differently shaped glycans on a cell (e.g., to a human cell glycan rather
than to a bird cell glycan; see Box 3-1). Thus, a remarkably small change
in the ability of a protein to bind to a specific linkage of a single monosac-
charide on cell surfaces can have a huge effect on society.
BOX 3-1
Pandemic Influenza
Four major human pandemics--in 1918, 1957, 1968, and 2009--were due to
influenza viruses from birds and swine crossing into the human population, caus-
ing widespread disease because of a lack of preexisting immunity. Concern about
pandemics from new viruses, such as the highly pathogenic H5N1 avian flu virus,
has drawn increased attention to the potential for influenza to cross species' barri-
ers. The designation H5N1 is a classification based on two proteins on the surface
of the virus that interact with host receptors. The H stands for hemagglutinin, which
attaches the virus to sialic acid receptors on cells. The N stands for neuraminidase,
which cleaves sialic acids to allow release of newly formed virus from the infected
cell. The neuraminidase is the target for current antiinfluenza medicines such as
Tamiflu and Relenza, which blocks the cycle of influenza replication.
Over 60 years ago influenza virus was found to bind to sialic acids on host
cells (Karlsson 1998). It is now known that the hemagglutinin of avian flu viruses
recognizes sialic acid receptors that differ from those recognized by human viruses.
Avian viruses recognize 2-3 linked sialic acids found in susceptible cells in birds,
while human influenza viruses recognize 2-6 linked sialic acids, which are found
in human airway cells (Stevens et al. 2006; Viswanathan et al. 2010). This small
difference is crucial for the transmission of influenza virus in humans. As a result,
receptor specificity represents a barrier for transmission of new animal viruses to
humans (Tumpey et al. 2007). Owing to the importance of receptor specificity, it is
now tracked by the Centers for Disease Control and Prevention as a risk factor for
the emergence of new human pandemics from animal influenza viruses.
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 45
FIGURE 3-2 The glycan shield of HIV. Glycans (blue) cover the HIV gp120 pro-
tein, which is responsible for virus binding to the CD4 receptor on T cells. The
binding and entry of HIV result in infection of the T cells and, ultimately, in
immunodeficiency. Figure 3-2
SOURCE: William Schief, The Scripps Research Institute, used with permission.
Bitmapped
The human immunodeficiency virus (HIV) that causes AIDS has had
an enormous impact on human health worldwide. The HIV coat protein
(Env) is among the most heavily glycosylated proteins known. As with
many viruses, the glycans are assembled using the host glycosylation
machinery (Raska and Novak 2010). The HIV virus uses this "glycan
shield" to prevent attack by the human immune system (see Figure 3-2).
However, recent findings show that humans immune to HIV produce
antibodies that bind to the glycan shield and neutralize infection by most
HIV strains (McLellan at al. 2011; Pejchal et al. 2011; Walker et al. 2011).
These insights are providing new hope for developing vaccine strategies
to prevent the disease. The glycans covering pg120 obscure it from recog-
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46 TRANSFORMING GLYCOSCIENCE
nition by the host immune system. The most potent antibodies identified
to date that neutralize HIV infection are those that bind to the glycan
shield.
Glycan-containing vaccines were first reported as early as 1929 (Tillett
and Francis 1929). Some of the most effective vaccines against infectious
organisms are directed toward glycans, and successful glycan-based vac-
cines include those against Haemophilus influenza and Streptococcus pneu-
moniae; others are in development. As of 2010, more than 30 glycan-based
vaccines were in preclinical and clinical trials (Astronomo and Burton
2010). Recent advances in glycomics, glycan synthesis, glycan arrays, and
methods for structural determination have resulted in a quantum leap
in glycan vaccine development (Seeberger and Werz 2007; Huang and
Wun 2010; Lepenies and Seeberger 2010). Advances in synthetic glycan
chemistry are also allowing researchers to create fully synthetic vaccines,
a development that may eliminate some safety concerns (Huang and Wun
2010). Still, many issues and challenges remain, such as the identification
of epitopes on glycans as a function of a pathogen's life cycle, stimulation
of both humoral and cellular immunity without triggering tolerance, and
the design of antigen presentation to generate high-avidity neutralizing
antibodies.
More than 15 million deaths occur annually because of parasitic dis-
eases. Even though the immune responses to parasites are almost always
directed against their unusual glycans (Nyame et al. 2004), there are
no effective vaccines against major parasitic diseases such as malaria,
trypanosomiasis, schistosomiasis, and amebiasis. Given the success of
glycan-based vaccines against bacteria and the growing knowledge with
respect to novel lineage-specific glycans in parasites, this area also repre-
sents a promising target for future vaccine development
While only a few examples are given here, it is clear that glycans
play a central role in our battle against invading organisms of all types.
As antibiotic resistance continues to rise, and the need for antivirals and
antiparasitics becomes acute, more focused efforts to understand the cen-
tral roles of glycans in infectious disease and vaccine development will
be increasingly important.
3.1.4 Glycans' Multifaceted Role in Cardiovascular Disease
Cardiovascular disease is the leading cause of death worldwide
(Nieuwdorp et al. 2005; Broekhuizen et al. 2009). It has recently become
clear that the glycocalyx of vascular endothelial cells plays a critical role
in the etiology of cardiovascular disease. This glycocalyx is comprised of
membrane glycoproteins, proteoglycans such as syndecans, and associ-
ated glycosylated plasma proteins. The molecules hyaluronan and hepa-
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 47
ran sulfate glycosaminoglycans are major components of the endothelial
glycocalyx. Normally, these glycans protect the vasculature from dam-
age, but disruption or damage of the endothelial glycocalyx contributes
directly to the onset of atherogenesis. The endothelial glycocalyx regu-
lates important enzymes such as nitric oxide synthase and superoxide
dismutase and serves as a barrier to macromolecules. In noninflamma-
tory states it also prevents the adherence of platelets and leukocytes
(Nieuwdorp et al. 2005).
Diabetes, another chronic disease of increasing prevalence and con-
cern, is a major cause of atherosclerosis. One current model suggests that
abnormal expression of proteoglycans or glycosaminoglycans in diabetics
contributes to the binding of cholesterol-rich lipoprotein particles at sites
in the vascular endothelium. Localized inflammation results in infiltration
of macrophages mediated by selectins, which then take up the lipopro-
tein particles to become foam cells, leading to a plaque that eventually
occludes the blood vessel (Tannock and King 2008). In contrast, heparan
sulfate proteoglycans play an important role in the clearance of lipopro-
tein particles from circulation by the liver (Stanford et al. 2009). Nuclear
and cytoplasmic protein glycosylation by the molecule O-GlcNAc also
plays an important role in diabetic cardiomyopathy (Clark et al. 2003;
Jones 2005; Fulop et al. 2007). Recent studies show that the contractile
machinery in the heart is heavily modified by O-GlcNAc and that both the
modification and association of O-GlcNAc cycling enzymes are strikingly
increased in diabetes (Ramirez-Correa et al. 2008). Both the glycocalyx
of endothelial cells and the modification of contractile machinery and
transcription factors regulating the expression of key cardiac proteins
represent novel targets for therapeutic discovery.
3.1.5 Glycans and the Molecular Mechanisms of Chronic Diseases
The prevalence of chronic diseases such as diabetes and Alzheimer's
disease is on the rise, and glycans appear to play critical roles in the etiol-
ogy of these and other chronic illnesses. Hyperglycemia and hyperlipid-
emia are the root cause of the biochemical events leading to the morbidity
and mortality associated with diabetes, and glycans are involved in regu-
lating a variety of cellular processes. Within the nucleus and cytoplasm,
modification of proteins by glycans is surprisingly abundant (Hart et
al. 2007; Copeland et al. 2008; Hart et al. 2011). Throughout the cell a
system for glycosylating proteins with the monosaccharide O-GlcNAc
has extensive cross talk with protein phosphorylation to regulate many
cellular processes in response to nutrients and stress, including transcrip-
tion, signaling, and cell division. Recent data from several laboratories
indicate that hyperglycemia-induced increases in O-GlcNAc modification
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60 TRANSFORMING GLYCOSCIENCE
inhibit the escape of soluble products, which in turn would slow hydro-
lysis rates. Thanks to 1 billion years of evolution, microbes have surfaces
that may enable them to disrupt this water layer, effectively solubilize the
plant cell wall, and capture solubilization products. Molecular mechanics
simulations appear to support this hypothesis (Himmel et al. 2007; Ding
et al. 2008; Lynd 2010). Manipulating or modifying such microbes may
lead to the generation of more effective microorganisms for consolidated
bioprocessing (Olson et al. 2011).
Another challenge in creating biofuels from plant biomass is to
increase production of biomass per hectare and to increase the yield
of fermentable sugars from each ton of biomass. Increased production
can be done by choosing the right crop for a given set of environmental
conditions and by increasing the resource efficiency of these plants by
decreasing water and fertilizer needs (Tilman et al. 2009; Lynd and Cruz
2010). From a biological perspective, one possibility is delaying the time
when plants flower. Keeping plants in a juvenile stage will also increase
biomass production. At least one research team has found that introduc-
ing a micro-RNA into a plant that caused it to remain in its juvenile stage
increased biomass production but also caused the plant to reduce lignin
production, leaving the resulting biomass more susceptible to degrada-
tion and conversion (Chuck et al. 2011).
3.2.3 Key Messages on Glycoscience and Energy
There is increasing interest in how to make use of the carbon in plant
biomass as an alternative source of energy to petroleum. However, plants
have evolved to resist destruction by the environment and by microbes,
and so efficient deconstruction of cell walls into carbohydrates and sugar
intermediates that can be fermented into biofuels is a challenge. Under-
standing how plant cell walls are synthesized and constructed and, con-
versely, how they can be effectively broken down remains a major goal
for improving biomass-derived energy. Addressing this challenge will
require continued research into the cell wall formation and breakdown
process and forms an important part of the glycoscience field.
As a result, the committee finds that:
· Plant cell walls, made mostly of glycans, represent the planet's
dominant source of biological carbon sequestration, or biomass,
and are a potentially sustainable and economical source of non-
petroleum-based energy.
· Understanding cell wall structure and biosynthesis and overcom-
ing the recalcitrance of plant cell walls to conversion into feed-
stocks that can be transformed into liquid fuels and other energy
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 61
sources will be important to achieving a sustainable energy revo-
lution. Glycoscience research will be necessary to advance this
area.
· Glycoscience can also contribute significantly to bioenergy devel-
opment by advancing the understanding of how to increase bio-
mass production per hectare and how to increase the yield of
fermentable sugar per ton of biomass.
3.3 GLYCOSCIENCE AND MATERIALS
As discussed above, plant cells walls represent the planet's dominant
biological carbon sequestration system. They are estimated to account for
120 billion to 170 billion tons per year. In comparison, the annual global
production of chemicals, including fertilizers but not pharmaceuticals,
was 1.2 billion tons in 2010. In other words, it takes plants three days to
produce biomass in the form of cell walls equal to the total annual output
of the world's chemical industry (DOE 2011).
Many of today's most widely used materials are petroleum based.
With the world's population growing, consumer-based economies
expanding, and petroleum resources ultimately being stretched in many
directions, there is a global need for the development of renewable and
sustainable resources that can serve as the source of a new generation of
materials. This is a surmountable task, one that could be at least partly
accomplished using polysaccharides produced by a wide variety of living
species, including plants, algae, fungi, and even insects and arthropods.
This diverse collection of organisms produces sufficient quantities of har-
vestable polysaccharides to potentially meet the world's demand both for
energy and synthetic materials (Perlack et al. 2005). Investigating glycan-
based materials further opens the possibility of designing new materials
with tailored properties that can expand on the range of materials cur-
rently available and may find new applications.
Materials based on polysaccharides, particularly cellulose-based poly-
saccharides, have a long history as a source of functional materials used
by human society. Wood, cotton, linen, hemp, and other cellulose-based
polysaccharides have been used as engineering materials for thousands
of years, and their use continues today, as evidenced by the enormity of
the worldwide industries in forest products, paper, and textiles, among
others. As a chemical raw material, cellulose has been used for about 150
years (Klemm et al. 2005). Regenerated cellulose has been used in the pro-
cessing of synthetic cellulose films (cellophane) and fibers (rayon), while
cellulose derivatives, made, for example, by replacing hydroxyl groups
with other functional groups, have been used to produce a wide variety of
cellulosic polymers, including cellulose esters and cellulose ethers, which
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62 TRANSFORMING GLYCOSCIENCE
have many industrial and pharmaceutical applications. Commercial
products made from cellulose derivatives include coatings, inks, binders,
thickening and gelling agents, and controlled-release drug tablets (Klemm
et al. 2005). Recently, the availability of cellulose-based nanoparticles has
begun changing the paradigm of what is achievable with natural materi-
als, including the production of biodegradable transparent films that are
stronger than steel (Moon et al. 2011). For two examples, see Box 3-3.
Further advances in glycoscience could drive the discovery of a wide
range of new sustainably produced polysaccharide-based materials in
three general categories: fine chemicals and feedstocks, polymeric materi-
als, and nanomaterials. Glycoscience has a real opportunity to positively
impact the progress of the baseline technologies needed to develop cost-
effective materials from polysaccharides that can compete, in terms of
cost and performance, with petroleum-based chemicals and polymers.
This vision can be achieved by providing mechanisms to develop an
increased understanding of polysaccharide biosynthesis in plants and
trees, new characterization tools and methods for understanding polysac-
charide structure, new methods for polysaccharide isolation, synthetic
process and chemical modification, and improved predictive modeling
capabilities.
3.3.1 Fine Chemicals and Feedstocks
A variety of polysaccharides are being investigated in the produc-
tion of functional chemical precursors that are then subsequently used to
make industrially relevant chemicals and engineering polymers (Bozell
and Petersen 2010). Examples of functional chemical precursors include
alcohols, such as ethanol, propanol, butanol, xylitol, and sorbitol; furans,
such as furfural and hydroxymethylfurfural; biohydrocarbons, such as
isoprene and long-chained hydrocarbons; and organic acids, such as lac-
tic acid, succinic acid, and levulinic acid. Carbohydrates are one of a
handful of natural products that can be used for production of many
chiral compounds with defined stereochemistries. Research has focused
on optimization of the bioconversion of polysaccharides in terms of yield,
rate, separation, titer, and product specificity. Much of this work has
focused on identifying and engineering improved fermentation organ-
isms, fermentation processes, and catalysts for converting sugars into
chemical precursors (Bozell and Petersen 2010). Significant advances will
require further development of methods to improve our ability to engi-
neer specific enzymes and organisms, such as yeast and fungi to produce
large yields of single materials. Advances in materials development will
also require a better understanding of how to achieve specific chemical
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 63
BOX 3-3
Examples of Carbohydrate-Based Materials:
Flexible Displays and Artificial Blood Vessels
Polysaccharides can be used in the development of a wide range of new ma-
terials with very diverse properties. For example, cellulose nanocomposites from
wood can be used as the basis for flexible, optically transparent materials that can
be used as substrates in the creation of luminescent organic light-emitting diodes.
Such materials have the potential for new applications in flexible electronics and
displays. Cellulose, in this case derived from bacteria, can also be used to make
thin, flexible tubes for use as implanted blood vessels.
Box 3-3 left
Glycan-based materials have multiple uses. SOURCES: Top, Reprinted from Okahisa et al.
2009, with permission from Elsevier; Botttom, Klemm et al. 2011, used with permission.
Box 3-3 right
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64 TRANSFORMING GLYCOSCIENCE
reactions with organisms and catalysts in a way that enables selective
reductions, conversions, and chemical bond formation or bond breaking.
3.3.2 Polymeric Materials
As discussed earlier, polysaccharides represent a broad range of natu-
ral polymers consisting of repeating sugar monomer units joined together
by glycosidic bonds. They can be linear or highly branched and can have
a wide variety of side groups. As a result of the variety of polysaccharides
produced in living species through their biosynthesis processes, the diver-
sity of polysaccharide materials available in nature creates an almost lim-
itless range of possibilities for creating useful new materials. For example,
trees produce cellulose and hemicelluloses, whereas nonwoody plants
produce cellulose, pectins, and starches, and various bacteria can synthe-
size polysaccharides that include glycogen, alginate, xanthan, dextran,
curdlan, gellan, colanic acid, K30 antigen, hyaluronic acid, and cellulose
(Rehm 2010). Each of these polysaccharides has a unique chemical struc-
ture comprising different combinations of sugars linked together in dif-
ferent configurations, all of which influence the properties of the given
polysaccharide in terms of its structural configuration, thermal stability,
reactivity, rheology, and mechanical properties.
With these natural materials as starting points, extensive research
has gone into the development of new reaction pathways that modify
the existing polysaccharide backbone structure or the side groups that
branch off the backbone. The goal of these efforts is to create new poly-
meric materials with novel properties and functions (Klemm et al. 2005;
Roy et al. 2009). For example, cellulose can be dissolved and the glu-
can chains reassembled to produce regenerated cellulose, or cellulose II,
which has been used to produce cellulose films, known as cellophane,
and fibers including rayon. Research has also yielded reaction pathways
that create cellulose derivatives such as cellulose acetate, cellulose acetate
propionate, cellulose acetate butyrates, carboxymethyl cellulose, cellulose
butyrate succinate, and cellulose acetate propionate (Klemm et al. 2005).
Each of these cellulose-based polymers was created by replacing acces-
sible hydroxyl groups with other chemical groups to produce a material
with novel performance characteristics. Given the wide range of polysac-
charides produced by nature, these examples represent only a fraction of
the potentially novel and useful materials that could be produced. But the
complexity and variability of polysaccharides represent a challenge that
requires fundamental research to develop:
· faster and more accurate methods for structural characterization
of polysaccharides,
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 65
· improved technology for separating and isolating polysaccha-
rides from their natural sources,
· novel chemical reactions to target chemical modifications to spe-
cific locations on the polysaccharides in order to create regioselec-
tive functionalization, and
· synthesis pathways for long-chain polysaccharides.
3.3.3Nanomaterials
Polysaccharides with linear or minimally branched backbone struc-
tures can self-assemble into ordered bundles in which the polymer chains
stack in parallel with each other along the chain axis. Such parallel-
stacked chains from a crystalline structure that can be characterized by
x-ray, neutron, and electron scattering techniques, among others. Two
primary examples of polysaccharides that show this crystalline structural
behavior are cellulose and chitin, a polysaccharide isolated from fungi
and from the exoskeleton of crustaceans and insects. The structures of
cellulose and chitin are similar, the difference being that chitin has one
hydroxyl group on each sugar replaced with an acetyl amine group.
However, the two polysaccharides have considerably different physical
and mechanical properties. Nonetheless, during the biosynthesis process,
both of these linear polysaccharides form fibril structures containing both
crystalline and amorphous arrangements of polymer chains. These fibrils
serve as the base reinforcement unit that provides the high mechanical
strength, strength-to-weight ratio, and toughness of plants, trees, crusta-
ceans, and insects.
Using specialized chemical-mechanical extraction methods, these
fibril structures and their crystalline regions can be isolated and used to
develop the next generation of plastics. To date, most work has been com-
pleted with cellulose, largely because of its availability and the extensive
scientific and technological expertise developed by the pulp and paper
industry. With this in mind, the next sections focus on cellulose to illus-
trate some of the opportunities for creating such nanomaterials, along
with the associated challenges. The issues discussed are relevant to other
ordered polysaccharides as well.
For cellulose the particles that are isolated after chemical and mechan-
ical extraction have dimensions on the nanoscale and are generically
called cellulose nanomaterials (CNs) (see Figure 3.4). CNs can have either
a rod and whiskerlike structure or a fibril particle morphology, with the
dimensions varying depending on cellulose source, extraction methods,
and extraction conditions (Habibi et al. 2010; Moon et al. 2011). Typically,
CNs range from 3 to 30 nanometers (nm) in diameter and from 50 nm to
several microns (µm) in length, with length-to-width (aspect) ratios of 20
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66 TRANSFORMING GLYCOSCIENCE
Figure 3-4
Bitmapped
FIGURE 3-4 Cellulose nanocrystals. Top, Stacking of cellulose chains showing
areas of "order" and "disorder." During one type of cellulose nanomaterial extrac-
tion process that uses acid hydrolysis, the amorphous regions are preferentially
dissolved and only the crystalline regions are left. Bottom, Transmission electron
Figure 3-4
micrograph of cellulose nanocrystals produced by acid hydrolysis.
SOURCE: Moon et al. 2011, used with permission.
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 67
to 100. CNs have high stiffness, low density, low thermal expansion, and
thermal stability up to about 200 to 300oC, and their surfaces can be read-
ily modified using a variety of chemical methods.
The sources from which CNs are extracted are themselves sustain-
able, biodegradable, and carbon neutral, and they generally have low
environmental, health, and safety risks. CNs have the potential to be pro-
cessed at industrial-scale quantities at low cost, although reaction condi-
tions, feedstock crystallinity, and other factors influence achievable yield
(Habibi et al. 2010; Qua et al. 2011). Preliminary testing shows minimal
environmental, health, and safety risks for CNs (Vartiainen et al. 2011),
although investigations into the environmental and health effects of all
types of nanoparticles continue to be an area of research and discussion.
With these advantages in mind, CNs are being considered for use in
the development of a variety of new plastics and composite structures,
including films, fibers, aerogels, and hydrogels. These materials have
potential applications in barrier films, separation membranes, antimicro-
bial films, transparent films, flexible displays, cardiovascular implants,
wound and burn dressings, tissue regeneration scaffolding, drug delivery
vehicles, fibers and textiles, templates for electronic components, batter-
ies, supercapacitors, electroactive polymers, and body armor.
Glycoscience could have a transformative impact on the CN bioplas-
tics industry by advancing the technologies necessary to control the crys-
talline structure, properties, surface chemistry, particle morphology, and
particle size distribution of CNs. Such technologies could then be used to
produce "tailored" CNs to meet specific performance metrics at low cost.
This vision can be achieved with advances in the following areas, each of
which are described in the sections that follow:
· understanding cellulose nanomaterial extraction processes,
· CN characterization,
· atomistic modeling of cellulose, and
· cellulose synthesis.
3.3.3.1 Understanding cellulose nanomaterial extraction processes
Unlike the extractions generally needed to produce fine chemicals,
feedstocks, and new polymers, the techniques needed to extract com-
pletely ordered polysaccharide particles from natural cellulose source
materials demand particular delicacy, and developing suitable processing
technologies requires new techniques and methodologies. Extracting CNs
from cellulose requires several steps, each of which influences particle
morphology or shape, particle size and size distribution, and interfacial
properties (Moon et al. 2011). Current processing technologies afford min-
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68 TRANSFORMING GLYCOSCIENCE
imal control of each of these physical properties, which in turn affects the
performance traits of the resulting nanoparticles. Because they are derived
from cellulose, a better understanding of the mechanisms involved in
plant cell wall construction and destruction can help provide insight into
new mechanisms that could be exploited to improve CN extraction meth-
ods to decrease the internal damage in CNs, narrow the particle size range
for a given CN processing methodology, improve extraction efficiencies,
increase CN yields, and scale production to industrial quantities.
3.3.3.2 Cellulose nanomaterial characterization
In addition to the structural and chemical characterization needed
to produce fine chemicals, feedstocks, and new polymers, novel struc-
tural, chemical, and mechanical characterization techniques are needed
for ordered polysaccharides. In particular, there is a need for techniques
that can characterize the configuration of the parallel-stacked polysaccha-
ride chains found in CNs. The properties of any given CN depend on the
arrangement of the cellulose chains, defects in the ordering of chains, local
changes in the chemistry internal to the CN, and changes in the chemistry
on the external CN surface. Characterizing these aspects is important for
understanding CN properties and for developing the means to rationally
design new CNs with specific properties. In addition, improved charac-
terization of structure, nanomechanical properties, and surface chemistry
will provide the opportunity to better understand processing-structure-
property relationships as they relate to the CN particles themselves and
to CN-CN and CN-water interactions, all of which are important for the
design of composite materials with improved performance.
While the cellulose polymorph structures of CNs are generally known,
characterization of individual CNs is currently lacking in such areas as
the percentage of crystallinity, the location of amorphous regions on the
CN surface or throughout the CN core, the fraction of a given cellulose
polymorph structure and its location in the particle, the identification
of defects such as missing cellulose chains, and the hydrogen bonding
networks both within and between cellulose chains in the CN. Character-
izing the elastic and tensile strength and other nanomechanical properties
of CNs remains a huge challenge, because their small size pushes the
limits of sensitivity of current methodologies such as atomic force micros-
copy (Wagner et al. 2011). While the reported properties for CNs are on
par with atomistic model predictions, these models are too variable for
conducting fundamental research on structure-property relationships, let
alone for assessing the influence of different extraction processes or cel-
lulose source on the quality of the resulting nanomaterial. Also lacking is
the ability to pinpoint the particular location of a given chemically func-
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GLYCOSCIENCE IN HEALTH, ENERGY, AND MATERIALS 69
tionalized side group along the CN surface. This information is important
to assess the accessibility of the given side group and how these groups
can interact with neighboring CNs or with the surrounding liquid in CN-
liquid suspensions.
3.3.3.3 Atomistic models of cellulose
New methods for atomistic modeling can contribute to better under-
standing of CN properties by providing insights into how to characterize
and describe the configuration of the parallel stacking of the polysaccha-
ride chains and how that configuration affects assembly properties. When
linked with experiments for direct comparison and validation, atomistic
modeling is a useful tool to help develop a better understanding of the
structure, mechanical properties, and surface chemistry of nanomaterials
such as CNs. Atomistic modeling can also provide information on the
interactions of CNs with neighboring nanoparticles, which affects the
composite properties of a CN-based material, and the interactions within
a particle suspension, which affects rheology. In addition, atomistic mod-
els that could accurately represent surface interactions and preferential
bonding site location would provide insights into composite properties
and aid efforts to tailor CN properties.
3.3.3.4 Cellulose synthesis
Today, it is particularly challenging to synthesize ordered polysac-
charides with the desired surface chemistry that self-assembles with
controlled crystalline properties. Through detailed understanding of
the polysaccharide biosynthesis process, it will ultimately be possible
to develop processing routes that facilitate tailoring both the side-group
chemistries of the polysaccharide backbone structure and the assembly of
individual chains into an ordered chain structure.
3.3.4 Key Messages on Glycoscience and Materials
Plastics and many other materials are currently derived from petro-
leum resources. Carbohydrates derived from plants, microorganisms,
insects, and other biological sources can provide a wealth of new options
for materials with diverse chemical and physical properties. Glycoscience
knowledge is helping unlock the development of these new materials
and the control of their design. The example of cellulose nanoparticles
was described in greater detail previously in this section to help illustrate
some of the research questions involved in characterizing and designing
new materials derived from carbohydrates. However, the need to under-
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70 TRANSFORMING GLYCOSCIENCE
stand chemical structures and interactions, to characterize and modify
materials, and to control production processes is important to develop-
ing functional and economically feasible new materials and products
more broadly. The application of glycoscience to materials science and
engineering represents an expanding area for the field and one for which
continued research and development will be needed.
As a result, the committee finds that:
· By fostering a greater understanding of the properties of gly-
cans and of plant cell wall construction and deconstruction,
glycoscience can play an important role in the development of
nonpetroleum-based sustainable new materials.
· Glycan-based materials have wide-ranging uses in such areas
as fine chemicals and feedstocks, polymeric materials, and
nanomaterials.
· There are many pathways to create a variety of functionalities on
a glycan, creating a wide range of options for tailoring material
properties.
3.4SUMMARY
This chapter explores three significant ways in which carbohydrates
contribute to society--in human health, in energy, and in materials. Gly-
cans are closely linked to both normal physiological function and to the
genesis and development of disease. They play promising roles in the dis-
covery of new diagnostic biomarkers for such diseases as cancer and for
new therapeutic targets. But glycans and glycoscience also have impor-
tant roles to play in the improved conversion of biofuels and the design
and creation of new carbohydrate-based materials. As a result, glycosci-
ence knowledge will contribute to the development of new energy and
materials science solutions that can replace some of the roles currently
played by petroleum-based products. This chapter attempts to provide a
sense of some of the unanswered questions in glycoscience as well as the
exciting potential that may be realized through future research. The next
chapter continues this discussion by exploring a set of questions that we
may not yet have the necessary tools to fully address but the pursuit of
whose answers remains an exciting challenge.
