Lehninger Principles of Biochemistry

1
Chap. 7B. Carbohydrates and Glycobiology

• Monosaccharides and Disaccharides
• Polysaccharides
• Glycoconjugates Proteoglycans, Glycoproteins,
  and Glycosphingolipids
• Carbohydrates as Informational Macromolecules
  the Sugar Code
• Working with Carbohydrates

Fig. 7-34. Helicobacter pylori cells adhering to
the gastric surface.
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Intro. to Glycoconjugates
In addition to their roles as energy storage and
structural polymers, polysaccharides and
oligosaccharides are information carriers. Some
provide communication between cells and their
extracellular surroundings others label proteins
for transport to and localization in specific
organelles, or for destruction when the protein
is malformed or superfluous and others serve as
recognition sites for extracellular signal
molecules (growth factors, for example) or
extracellular parasites (bacteria and viruses).
On almost every eukaryotic cell, specific
oligosaccharide chains attached to components of
the plasma membrane form a carbohydrate layer
(the glycocalyx), several nanometers thick, that
serves as an information-rich surface that the
cell presents to its surroundings. These
oligosaccharides are central players in cell-cell
recognition and adhesion, cell migration during
development, blood clotting, the immune response,
wound healing, and other cellular processes. In
most of these cases the informational
carbohydrate is covalently joined to a protein or
a lipid to form a glycoconjugate, which is the
biologically active molecule.
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Glycoconjugate Classes
Three types of glycoconjugates occur in nature
(Fig. 7-24). Proteoglycans are macromolecules of
the cell surface or extracellular matrix in which
one or more sulfated glycosaminoglycan chains are
joined covalently to a membrane protein or a
secreted protein. Proteoglycans are major
components of all ECMs. Glycoproteins have one or
several oligosaccharides of varying complexity
joined covalently to a protein. They are usually
found on the outer face of the plasma membrane as
part of the glycocalyx, in the ECM, or in the
blood. The oligosaccharide portions of
glycoproteins are very heterogeneous and are rich
in structural information. Some cytosolic and
nuclear proteins can be glycosylated as well. The
glycosphingolipids are plasma membrane lipids in
which the hydrophilic head groups are
oligosaccharides. Both glycoproteins and
glycosphingolipids are recognized and bound by
carbohydrate-binding proteins called lectins.
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Structure of Proteoglycans (I)
Mammalian cells can produce 40 types of
proteoglycans. These molecules act as tissue
organizers, and they influence various cellular
activities, such as growth factor adhesion and
activation. The basic proteoglycan unit consists
of a core protein to which glycosaminoglycan(s)
are covalently attached. The point of attachment
is a serine residue, to which the
glycosaminoglycan is joined through a
tetrasaccharide bridge (Fig. 7-25). The Ser
residue is generally in the sequence
-Ser-Gly-X-Gly-, where X is any amino acid. The
xylose residue at the reducing end of the bridge
is joined by its anomeric carbon to the hydroxyl
of the Ser residue.
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Structure of Proteoglycans (II)
Many proteoglycans are secreted into the ECM, but
some remain bound to the cell membrane of origin
as integral membrane proteins. Two major families
of membrane-bound heparan sulfate proteoglycans
are described in Fig. 7-26a. The syndecans have a
single transmembrane domain and an extracellular
domain bearing three to five chains of heparan
sulfate and in some cases chondroitan sulfate.
Glypicans are attached to the membrane via a
lipid anchor, which is a derivative of the
membrane lipid phosphatidylinositol. Both
syndecans and glypicans can be shed into the
extracellular space after cleavage by a protease
and a phospholipase, respectively, bound to the
ECM. Proteoglycan shedding is involved in
cell-cell recognition and adhesion, and in
proliferation and differentiation of cells.
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Structure of Proteoglycans (III)
The glycosaminoglycan chains can bind to a
variety of extracellular ligands and thereby
modulate the ligands interaction with specific
receptors on the cell surface. Glycosaminoglycans
such as heparan sulfate contains domains
(typically 3 to 8 disaccharides long) that differ
from neighboring domains in sequence and in their
ability to bind to specific proteins. Highly
sulfated domains (called NS domains) alternate
with domains having unmodified residues (NA
domains, for N-acetylated domains) (Fig. 7-26b).
The exact pattern of sulfation in the NS domains
depends on the particular proteoglycan.
Furthermore, the same core protein can display
different heparan sulfate structures when
synthesized in different cell types.
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Examples of Proteoglycan Functions (I)
Heparan sulfate molecules with precisely
organized NS domains bind specifically to
extracellular proteins and signaling molecules to
alter their functions. In the first example (Fig.
7-27a), the binding of antithrombin (AT) to the
heparan sulfate moiety of a proteoglycan in the
ECM alters the conformation of AT so that it
binds to the blood clotting factor Xa, preventing
clotting. AT recognizes a specific sulfated NS
domain in heparan sulfate.
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Examples of Proteoglycan Functions (II)
In another example of proteoglycan function (Fig.
7-27b), the binding of AT and thrombin to two
adjacent NS domains of heparan sulfate chains in
a proteoglycan brings them into close proximity,
where they can bind to one another, and AT
inhibits the activity of thrombin.
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Proteoglycan Aggregates
Some proteoglycans can form proteoglycan
aggregates, enormous supramolecular assemblies of
many glycosaminoglycan-decorated core proteins
all bound to a single molecule of hyaluronan
(Fig. 7-28). The core protein involved commonly
is aggrecan (Mr 250,000). Aggrecan binds multiple
chains of chondroitin sulfate and keratin sulfate
via covalent linkages to serine residues. When a
hundred or more of these decorated core proteins
binds a single extended hyaluronan molecule the
resulting proteoglycan aggregate has a mass of Mr
gt2 x 108. Its size is comparable to a bacterial
cell. Aggrecan interacts with collagen in the ECM
of cartilage, contributing to the development,
tensile strength, and resilience of this
connective tissue.
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Interactions Between Cells and the ECM
The ECM is a strong and resilient meshwork
containing proteoglycans in association with
collagen, elastin, and fibronectin. Some of these
proteins are multiadhesive, with a single protein
having binding sites for several different matrix
molecules. The ECM protein fibronectin, for
example, has several separate domains that bind
fibrin, heparan sulfate, collagen, and a family
of plasma membrane proteins called integrins.
Integrins mediate signaling between the cell
interior and the ECM. A diagram showing just some
of the interactions occurring between a cell and
the surrounding ECM is shown in Fig. 7-29. These
interactions not only anchor the cell in the ECM,
but also provide paths that direct the migration
of cells in developing tissue and convey
information in both directions across the plasma
membrane.
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Structure of Glycoproteins
Glycoproteins are carbohydrate-protein conjugates
in which the glycans are smaller, branched, and
more structurally diverse than the huge
glycosaminoglycans present in proteoglycans. Two
broad classes of glycoproteins occur in
nature--the O-linked and the N-linked
glycoproteins (Fig. 7-30). In O-linked
glycoproteins, the carbohydrate is attached at
its anomeric carbon through a glycosidic linkage
to the -OH group of a Ser or Thr residue. Both a
simple chain and a complex carbohydrate chain are
shown in the figure. In an N-linked glycoprotein,
the carbohydrate is attached via an N-glycosyl
linkage to the amide nitrogen of an Asn residue
of the protein. N-linked oligosaccharide chains
are attached at Asn residues that are part of the
consensus sequence -N-P-ST-, although not all
such sequences in proteins are modified. There
appears to be no specific consensus sequence for
the attachment of O-linked oligosaccharides.
Three types of N-linked oligosaccharide chains
that are common in glycoproteins are shown in the
figure. The mechanism of synthesis and attachment
of these chains is covered in Chap. 27.
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Functions of Glycoproteins
About half of all proteins of mammals are
glycosylated. Many of these are plasma membrane
proteins and secretory proteins. The external
surface of the plasma membrane has many membrane
glycoproteins with vast types of covalently
attached oligosaccharides of varying complexity.
Examples of glycosylated secretory proteins
include immunoglobulins (antibodies) and certain
hormones such as follicle-stimulating hormone and
luteinizing hormone. However, another class of
glycoproteins found in the cytoplasm and nucleus
carry only a single residue of N-acetylglucosamine
attached in O-glycosidic linkage to the hydroxyl
group of Ser side-chains. This modification is
reversible and often occurs on the same Ser
residues that are phosphorylated. The two
modifications are mutually exclusive, and the
modified protein exhibits different activity in
the two modified states. The biological
advantages of protein glycosylation include
improving the solubility of proteins, providing
address labels directing targeting in cells and
between tissues, and protein folding and
stabilization. At this time 18 different genetic
diseases of protein glycosylation have been
described in humans. Finally, the discipline of
glycomics, the systematic characterization of all
of the carbohydrate components of a given cell or
tissue, offers insight into the normal patterns
of glycosylation in cells and how they may be
perturbed in diseases such as cancer.
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Glycolipids and Lipopolysaccharides (I)
In glycolipids and lipopolysaccharides, complex
oligosaccharide chains are attached to
membrane-anchored lipid moieties. The complex
structure of the lipopolysaccharide found in the
outer membrane of Salmonella typhimurium is shown
in Fig. 7-31. These molecules, which are found in
other Gram-negative bacteria such as E. coli, are
recognized by the immune system of vertebrates
and therefore are important determinants of the
serotypes of bacterial strains. Serotypes are
strains that are distinguished on the basis of
antigenic properties. The fatty acid-containing
lipid A portion of lipopolysaccharide is called
endotoxin. Its toxicity to humans is responsible
for the dangerously lowered blood pressure that
occurs in toxic shock syndrome resulting from
Gram-negative bacterial infections.
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Glycolipids and Lipopolysaccharides (II)
Plants and animals contain many types of
glycolipids and glycosphingolipids. The
gangliosides are glycosphingolipids containing
complex oligosaccharide chains with sialic acid
residues. These lipids are important for
cell-cell recognition, and some serve as
receptors for the entry of bacterial toxins such
as cholera toxin into mammalian cells. Other
glycosphingolipids, the globosides, contain
oligosaccharide chains that serve as the blood
group antigens. The structures of several
glycosphingolipids are covered in Chap. 10.
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Carbohydrates as Informational Molecules The
Sugar Code
Glycobiology is the study of the structure and
function of glycoconjugates. Cells use specific
oligosaccharides to encode important information
about intracellular targeting of proteins,
cell-cell interactions, cell differentiation and
tissue development, and extracellular signals.
The oligosaccharides of glycoproteins and
glycolipids are highly complex and diverse.
Oligosaccharides of typical glycoproteins can
contain a dozen or more monosaccharide residues
in a variety of branched and linear structures,
and in a variety of linkages. (See the N-linked
oligosaccharides in the glycoprotein examples
shown in Fig. 7-30). With the assumption that 20
different monosaccharide subunits are available
for the construction of oligosaccharides, it can
be calculated that many billions of different
hexameric oligosaccharides, for example, are
possible. This compares with 6.4 x 107 (206)
different hexapeptides possible from the 20
common amino acids, and 4,096 (46) different
hexanucleotides with the four bases. The
structural information potentially present in
glycans thus actually surpasses that of nucleic
acids for molecules of modest size. Each of the
oligosaccharides attached to a glycoprotein for
example, presents a unique three-dimensional
structure--a word in the sugar code--that is
readable by the proteins that interact with it.
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Lectin Functions (I)
Lectins, found in all organisms, are proteins
that bind carbohydrates with high specificity and
with moderate to high affinity. Lectins play
roles in a wide variety of cell-cell recognition,
signaling, and adhesion processes and in
intracellular targeting of newly synthesized
proteins. Some examples of these functions are
covered in the next few slides. The first example
covered concerns the role of the
asialoglycoprotein receptor (a lectin) of the
liver which is important in the clearance of many
plasma glycoproteins from the circulation.
Normally, many plasma glycoproteins are
synthesized with oligosaccharide chains that
terminate with N-acetylneuraminic acid (a sialic
acid) (see figure). This sugar protects the
glycoproteins initially from uptake and
degradation by hepatocytes. However, during the
lifetime of the glycoprotein in the bloodstream,
its sialic
acid residues are gradually removed by enzymes
called neuraminidases (sialidases) of unknown
origin. These asialo- versions of the original
glycoproteins are then taken up by the liver and
degraded. Note that a similar mechanism is
apparently responsible for the removal of old
erythrocytes from the bloodstream by the spleen.
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Lectin Functions (II)
Several animal viruses, including influenza
virus, attach to their host cells though the
interactions of viral lectins (e.g., influenza
virus hemagglutinin protein) and oligosaccharides
displayed on the host cell surface. After the
virus has replicated inside the host cell, the
newly synthesized viral particles bud out of the
cell, wrapped in a portion of its plasma
membrane. A viral sialidase trims the terminal
sialic acid residues from the host cells
oligosaccharides, releasing the viral particles
from their interaction with the cell and
preventing their aggregation with one another.
Another round of infection can then begin. The
antiviral drugs oseltamivir (Tamiflu) and
zanamivir (Relenza) are used in the treatment of
influenza. These drugs are sugar analogs that are
structurally similar to sialic acid
(N-acetylneuraminic acid) (Fig. 7-33a). They
therefore can inhibit the viral sialidase and
limit the release of viruses from the original
infected cell.
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Lectin Functions (III)
The binding site on influenza neuraminidase
(sialidase) for N-acetylneuraminic acid and the
antiviral drug, oseltamivir (Tamiflu) is shown in
Fig. 7-33 b-d. Tamiflu competitively inhibits the
binding of terminal sialic acid residues of
oligosaccharide chains to the neuraminidase. When
Tamiflu binds to the active site it pushes away
the side-chain of Glu276, making room for the
binding of the inhibitor (Part c). Mutations in
viral neuraminidase, however, which replace
nearby His274 with a bulky Tyr residue, prevent
the repositioning of Glu276, and Tamiflu binding
to the active site (Part d).
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Lectin Functions (IV)
Some microbial pathogens have lectins that
mediate bacterial adhesion to host cells or the
entry of toxins into cells. For example, the
gastric ulcer-causing bacterium, Helicobacter
pylori, has a surface lectin that adheres to
oligosaccharides on the surface of epithelial
cells that line the inner surface of the stomach
(Fig. 7-34). This allows the bacterium to
colonize the stomach and cause associated
ulceration. The H. pylori lectin actually binds
to the Lewis b (Leb) antigen present in cell
surface glycoproteins and glycolipids that
defines the type O blood group. This observation
helps explain the several-fold greater incidence
of gastric ulcers in people of blood type O than
in those of type A or B. Chemically synthesized
analogs of the Leb antigen may prove useful in
treating this type of ulcer.
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Analysis of Glycoprotein Oligosaccharides
An overview of some of the procedures used to
analyze the total composition, linkages, and
sequences of monosaccharides in oligosaccharide
chains is presented in Fig. 7-38. The method
starts with the removal of oligosaccharide chains
from the glycoproteins that contain them by
enzymes called endoglycosidases. It typically
ends with the fine-structure analysis of the
chains by NMR and mass spectrometry.
Oligosaccharide structure analysis is more
complicated than protein and nucleic acid
analysis due to the branching and variety of
linkages present in these molecules.