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.
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.
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 carbohydratebinding proteins called lectins.
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.
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.
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.
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.
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.
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 >2 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.
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.
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.
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 Nacetylglucosamine 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.
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.
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.
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.
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.
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 cell’s
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. 733a). They therefore can inhibit the viral sialidase and limit the
release of viruses from the original infected cell.
Lectin Functions (III)
The binding site on influenza neuraminidase (sialidase) for Nacetylneuraminic 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).
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.
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.