2. Overview of glycosaminoglycans
• Glycosaminoglycan (GAGs) are large complexes of
negatively charged heteropolysaccharide chains. They
are generally associated with a small amount of protein,
forming proteoglycans, which typically consist of > 95%
CHO.
Note: this is in comparison to the glycoproteins, which
consist primarily of protein with a small amount of CHO
• Glycosaminoglycans have the special ability to bind large
amounts of water, thereby producing the gel-like matrix
that forms the basis of the body’s ground substance
• The viscous, lubricating properties of mucous secretions
also result from the presence of GAGs, which led to the
original naming of these cpds as mucopolysaccharides
3. II. Structure of GAGs
• GAGs are long, unbranched, heteropolysaccharide chains
generally composed of a repeating disacch unit [acidic sugar-
amino sugar]n
• The amino sugar is either D-glucosamine or D-galactosamine, in
which the amino group is usually acetylated, thus eliminating its
+ve charge. The amino sugar may also be sulfated on C-4 or 6 or
on a non-acetylated nitrogen
• The acidic sugar is either D-glucuronic acid or its C-5 epimer, L-
iduronic acid
Note: a single exception is keratan sulfate, in which galactose rather
than an acidic sugar is present
• These acidic sugars contain carboxyl groups that are negatively
charged at physiologic pH &, together with the sulfate groups,
give GAGs their strongly –ve nature
6. A. Relationship between GAG structure and function
- Because of their large # of –ve charges, these
heteropolysacch chains tend to be extended in solutions.
They repel each other and are surrounded by a shell of
water molecules.
- When brought together, they “slip” past each other, much
as two magnets with the same polarity seem to slip past
each other. This produces “slippery” consistency of
mucous secretions & synovial fluid
- When a soln of GAGs is compressed, the water is
“squeezed out” & the GAGs are forced to occupy a
smaller volume. When the compression is released, the
GAGs spring back to their original, hydrated volume
because of repulsion of their –ve charges. This property
contributes to the resilience of synovial fluid & the
vitreous humor of the eye.
8. B. Classification of GAGs
- The six major classes of GAGs are divided according to
monomeric composition, type of glycosidic linkages, &
degree & location of sulfate units.
- The structure of the GAGs & their distribution in the body
illustrated in Fig. 4.
9.
10. C. Structure of proteoglycans
- All of the GAGs, except hyaluronic cid, are found covalently
attached to proteins, forming proteoglycan monomers.
1. Structure of proteoglycan monomer:
- A proteoglycan monomer found in cartilage consists of a core protein
to which the linear GAG chains are covalently attached
- These chains which may each be composed of >100 monosacch,
extend out from the core protein & remain separated from each
other because of charge repulsion. The resulting structure
resembles a “bottle brush”
- In cartilage proteoglycan, the species of GAGs include chondroitin
sulfate & keratan sulfate
Note: a number of proteoglycans have been characterized & named
based on their structure & functional location. E.g., syndecan is an
integral memb proteoglycan, versican & aggrecan are the
predominant extracellular proteoglycans, & neurocan & cerebrocan
are found primarily in the NS.
12. 2. Linkage between the carbohydrate chain & the protein
- This linkage is most commonly through a trihexoside
(galactose-galactose-xylose) & a ser residue,
respectively. An O-glycosidic bond is formed b/w the
xylose & the hydroxyl group of the ser.
13. 3. Proteoglycan aggregates
- The proteoglycan monomers associate
with a molecule of hyaluronic acid to form
proteoglycan aggregates. The association
is not covalent, but occurs primarily
through ionic interactions b/w core protein
& the hyaluronic acid
- The association is stabilized by additional
small proteins called link proteins
15. III. Synthesis of glycosaminoglycans
- The polysacch chains are elongated by the
sequential addition of alternating acidic & amino
sugars, donated by their UDP-derivatives
- The reactions are catalyzed by specific
transferases
- The synthesis of the GAGs is analogous to that
of glycogen except that the GAGs are produced
for export from the cell. The synthesis occurs,
therefore, in the ER & the Golgi, rather than in
the cytosol.
16. A. Synthesis of amino sugars
- Amino sugars are essential components of GAGs, glycoproteins,
glycolipids, & certain oligosaccharides, & are also found in some
antibiotics. The synthetic pathway of amino sugars is very active in
connective tissues, where as much as 20% of gluc flows through the
pathway.
1. N-acetylglucosamine (gluNAc) & N-acetylgalactosamine (galNAc):
- The monosacch F-6-P is the precursor of gluNAc, galNAc, & the sialic
acids, including N-acetylneuraminic acid (NANA, a nine-carbon,
acidic monosacch).
- In each of these sugars, a hydroxyl group of the precursor is
replaced by an amino group donated by the aa, glutamine
Note: the amino groups are almost always acetylated
- The UDP-derivatives of gluNAc & galNAc are synthesized by
reactions analogous to those described for UDP-glucose synthesis.
These are activated forms of the monosaccharides that can be used
to elongate the CHO chain
17. 2. N-Acetylneuraminic acid:
- NANA is a member of the family of sialic acids, each of
which is acylated at a different site. These cpds are
usually found as terminal CHO residues of
oligosaccharide side chains of glycoproteins, glycolipids,
or, less frequently, of GAGs
- The carbons & nitrogens in NANA come from N-
acetylmannosamine & PEP
Note: before NANA can be added to a growing oligosacch,
it must be converted into its active form by reacting with
cytidine triphosphate (CTP). The enz N-
acetylneuraminate-CMP-pyrophosphorylase removes
pyrophosphate from CTP & attaches the remaining CMP
to the NANA. This is the only nucleotide sugar in human
metabolism in which the carrier nucleotide is a
monophosphate
19. B. Synthesis of acidic sugars
- D-glucuronic acid, whose structure is that of gluc with an
oxidized C-6 (-CH2OH → -COOH), & its C-5 epimer, L-
iduronic acid, are essential components of GAGs.
- Glucuronic acid is also required in detoxification
reactions of a number of insoluble cpds, e.g., bilirubin,
steroids, & several drugs.
- In plants & mammals (other than guinea pigs & primates,
including man), glucuronic acid serves as a precursor of
ascorbic acid (vitamin C).
- The uronic acid pathway also provides a mechanism by
which dietary D-xylulose can enter the central metabolic
pathways
20. 1. Glucuronic acid:
- Glucuronic acid can be obtained in small amounts from diet.
It can also be obtained from the intracellular lysosomal
degradation of GAGs, or via the uronic acid pathway.
- The end-product of glucuronic acid metabolism in humans is
D-xylulose 5-P, which can enter the hexose monophosphate
pathway & produce the glycolytic intermediates GA-3P & F-
6-P
- The active form of glucuronic acid that donates the sugar in
GAG synthesis & other glucuronylating reactions is UDP-
glucuronic acid, which is produced by oxidation of UDP-
glucose
2. L-iduronic acid synthesis:
- Synthesis of L-iduronic acid residues occurs after D-
glucuronic acid has been incorporated into the CHO chain.
- Uronosyl 5-epimerase causes epimerization of the D- to L-
sugar
23. C. Synthesis of the core protein
- The core protein is synthesized on & enters rER. The
protein is then glycosylated by memb-bound transferases
as it moves through ER.
D. Synthesis of the carbohydrate chain
- CHO chain formation begins by synthesis of a short
linkage region on the core protein on which CHO chain
synthesis will be initiated.
- The most common linkage region is formed by the transfer
of xylose from UDP-xylose to the hydroxyl group of a ser
(or thr) catalyzed by xylosyltransferase.
- Two galactose molecules are then added, completing the
trihexoside.
- This is followed by sequential addition of alternating acidic
& amino sugars, & conversion of some D-glucuronyl to L-
iduronyl residues
25. E. Addition of sulfate groups
- Sulfation of CHO chain occurs after the
monosacch to be sulfated has been
incorporated into the growing CHO chain
- The source of the sulfate is 3`-
phosphoadenosyl-5`-phosphosulfate (PAPS, a
molecule of AMP with a sulfate group attached
to the 5`-phosphate).
- Sulfotransferases cause the sulfation of the
CHO chain at specific sites.
Note: a defect in the sulfation process results in
one of several autosomal recessive disorders
that affect the proper development &
maintenance of the skeletal system. This
illustrates the importance of the sulfation step
26. IV. Degradation of glycosaminoglycans
- GAGs are degraded in lysosomes, which contain
hydrolytic enz’s that are most active at a pH of ~
5 [Note: therefore, as a group, these are called
acid hydrolases]
- The low pH optimum is a protective mechanism
that prevents the enz’s from destroying the cell
should leakage occur into cytosol where pH is
neutral.
- With exception of keratan sulfate, which has a
half-life of > 120 days, the GAGs have a
relatively short half-life, ranging from ~ 3 days
for hyaluronic acid to 10 days for chondroitin &
dermatan sulfate
27. A. Phagocytosis of extracellular glycosaminoglycans
- Because GAGs are extracellular or cell-surface cpds, they must be
engulfed by an invagination of the CM (phagocytosis), forming a
vesicle inside of which the GAGs are to be degraded.
- The vesicle then fuses with a lysosome, forming a single digestive
vesicle in which GAGs are efficiently degraded
B. Lysosomal degradation of GAGs
- The lysosomal degradation of GAGs requires a large # of
acid hydrolases for complete digestion.
- 1st , the polysacch chains are cleaved by
endoglycosidases, producing oligosaccharides. Further
degradation of the oligosacch’s occurs sequentially from
the non-reducing end of each chain, the last group
(sulfate or sugar) added during synthesis being the 1st to
be removed. Examples of some of these enz’s & the
bonds they hydrolyze Fig 12.
28. V. Mucopolysaccharidosis
- Mucopolysaccharidoses are hereditary disorders that are
clinically progressive. They are characterized by
accumulation of GAGs in various tissues, causing varied
symptoms, such as skeletal & extracellular matrix
deformities, & mental retardation.
- Mucopolysaccharidoses are caused by a deficiency of
one of the lysosomal hydrolases normally involved in
degradation of heparan sulfate and/or dermatan sulfate.
- This results in the presence of oligosacch’s in urine,
because of incomplete lysosomal degradation of GAGs.
These fragments can be used to diagnose the specific
mucopolysaccharidosis, namely by identifying the
structure present on the non-reducing end of oligosacch.
That residue would have been the substrate for the
missing enz.
29. - Diagnosis can be confirmed by measuring the patients
cellular level of lysosomal hydrolases. Children who are
homozygous for one of these diseases are apparently
normal at birth, then gradually deteriorate. In severe
cases, death occurs in childhood.
- All of the deficiencies are autosomal & recessively
inherited except Hunter syndrome, which is X-linked
- Bone marrow transplants are currently being used
successively to treat Hunter syndrome; the transplanted
macrophages produce the sulfatase needed to degrade
GAGs in the extracellular space.
Note: some of lysosomal enzymes required for degradation
of GAGs also participate in degradation of glycolipids &
glycoproteins. Therefore, an individual suffering from a
specific mucopolysaccharidosis may also have a
lipidosis or glycoprotein-oligosaccharidosis
30. Figure 14.12
Degradation of the
glycosaminoglycan
heparan sulfate by
lysosomal enzymes,
indicating
sites of enzyme
deficiencies in some
representative
mucopolysaccharidoses.
32. Facies of a male with the mucopolysaccharidosis, Hunter
syndrome
33. VI. Overview of glycoproteins
- Glycoproteins are proteins to which oligosacch’s are covalently attached.
They differ from proteoglycans (which might be considered a special case of
glycoproteins) in that length of glycoproteins’ CHO chain is relatively short
(usually 2-10 sugar residues in length, although they can be longer),
whereas it can be very long in the GAGs.
- In addition, whereas GAGs have diglucosyl repeat units, the CHO’s of
glycoproteins do not have serial repeats.
- The glycoprotein CHO chains are often branched instead of linear, & may or
may not be negatively charged.
- Glycoproteins contain highly variable amounts of CHO. e.g., the
immunoglobulin IgG, contains < 4% of its mass as CHO, whereas human
gastric glycoprotein (mucin) contains > 80% CHO
- Membrane-bound glycoproteins participate in a broad range of cellular
phenomena, including cell surface recognition (by other cells, hormones,
viruses), cell surface antigenicity (such as the blood group antigens), and as
components of the extracellular matrix & of the mucins of the
gastrointestinal & urogenital tracts, where they act as protective biologic
lubricants.
- In addition, almost all of the globular proteins present in human plasma are
glycoproteins
35. VII. Structure of glycoprotein oligosaccharides
- The oligosaccharide components of glycoproteins are
generally branched heteropolymers composed primarily
of D-hexoses, with the addition in some cases of
neuraminic acid, & L-fucose, a 6-deoxyhexose
A. Structure of the linkage between carbohydrate and
protein
- The oligosacch may be attached to the protein through an
N- or O-glycosidic link.
- In the former case, sugar chain is attached to the amide
group of an asparagine side chain, & in the latter case,
to a hydroxyl group of either ser or thr R-group.
Note: in case of collagen, there is an O-glycosidic linkage
b/w galactose or gluc & hydroxyl group of hydroxylysine
36. B. N- and O-linked oligosaccharides
- A glycoprotein may contain only one type of
glycosidic linkage (N- or O-linked), or may
have both O- and N-linked oligosacchs within
same molecule
1. O-linked oligosaccharides:
- The O-linked oligosacch’s may have one or
more of a wide variety of sugars arranged in
either a linear or branched pattern.
- Many O-linked oligosacch’s are found as
memb glycoprotein components or in
extracellular glycoproteins. E.g., O-linked
oligosacch’s help provide the ABO blood group
determinants
37. 2. N-linked oligosaccharides:
- The N-linked oligosacch’s fall into 2 broad
classes: complex oligosacch’s & high-
mannose oligosacch’s. both contain same core
pentasaccharide (Fig 14), but the complex
oligosacch’s contain a diverse group of
additional sugars, e.g., N-acetylglucosamine
(GlcNAc), L-fucose (Fuc), N-acetylneuraminic
acid (NANA), whereas the high-mannose
oligosacch’s contain primarily mannose (Man)
39. VIII. Synthesis of glycoproteins
- Most proteins are destined for the cytoplasm & are synthesized on
free ribosomes in cytosol. However, proteins, including many
glycoproteins, that are destined for cellular memb’s, lysosome, or to
be exported from cell, are synthesized on ribosomes attached to
rER.
- These proteins contain specific signal sequences at their N-terminal
end that act as molecular “address labels” which direct the proteins
to their proper destinations
- These signal sequences allow growing polyp to be extruded into
lumen of rER. The proteins are then transported via secretory
vesicles to Golgi complex, which acts as a sorting center.
- In Golgi those glycoproteins that are to be secreted from cell (or are
targeted for lysosomes) remain free in lumen, whereas those that
are to become components of the CM become integrated into Golgi
memb, their CHO portions oriented toward lumen.
- Vesicles bud off from Golgi & fuse with the CM, either releasing the
free glycoproteins, or adding the memb-bound proteins of the
vesicle to the CM. The memb glycoproteins are thus oriented with
the CHO portion on the outside of the cell.
40. Figure 14.15
Transport of glycoproteins
through the Golgi apparatus
and their subsequent
release or
incorporation into a
lysosome or the cell
membrane.
41. A. Carbohydrate components of glycoproteins
• The precursors of the CHO components of glycoproteins
are sugar nucleotides, which include UDP-glucose,
UDP-galactose, UDP-N-acetylglucosamine & UDP-N-
acetylgalactosamine. In addition, GDP-mannose, GDP-
L-fucose (which is synthesized from GDP-mannose), &
CMP-N-acetylneuraminic acid may donate sugars to the
growing chain.
Note: when NANA is present, the oligosacch has a –ve
charge at physiologic pH.
• The oligosacch’s are covalently attached to specific aa
R-groups of the protein, where the 3-D structure of the
protein determines whether or not a specific aa R-group
is glycosylated
42. B. Synthesis of O-linked glycosides
- Synthesis of O-linked glycosides is very similar to that of the GAGs.
1st the protein to which the oligosacch’s are to be attached is
synthesized on the rER, & extruded into its lumen.
- Glycosylation begins immediately, with the transfer of an N-
acetylgalactosamine (from UDP-N-acetylgalactosamin) onto a
specific seryl or threonyl R-group.
1. Role of glycosyltransferases
- The glycosyltransferases responsible for the stepwise synthesis of
the oligosacch’s are bound to the memb’s of the ER or the Golgi
apparatus.
- They act in a specific order, without using a template as is required
for DNA, RNA, & protein synthesis, but rather by recognizing the
actual structure of the growing oligosacch as the appropriate
substrate
43. C. Synthesis of the N-linked glycosides
• The synthesis of N-linked glycosides also occurs in the
lumen of the ER & in Golgi. However, these structures
undergo additional processing steps, & require the
participation of a lipid (dolichol) & its phosphorylated
derivative, dolichol pyrophosphate
1. Synthesis of dolichol-linked oligosaccharide
• 1st, as with O-linked glycosides, protein is synthesized on
rER & enters its lumen. The protein itself does not
become glycosylated with individual sugars at this stage
of glycoprotein synthesis, but rather a lipid-linked
oligosacch is 1st constructed
• This consists of dolichol (an ER memb lipid 80-100
carbons long) attached through a pyrophosphate linkage
to an oligosacch containing N-acetylglucosamine,
mannose, & glucose.
44. • The sugars to be added to the dolichol by the memb-bound
glycosyltransferases are first N-acetylglucosamine, followed by
mannose & gluc.
• The oligosacch is transferred from the dolichol to an asparagine side
group of the protein by a protein-oligosaccharide transferase present
in the ER
2. Final processing of N-linked oligosaccharides:
• After incorporation into the protein, the N-linked oligosacch is
processed by removal of specific mannosyl & glucosyl residues as
the glycoprotein moves through the ER
• Finally, the oligosacch chains are completed in Golgi by addition of a
variety of sugars (e.g., N-acetylglucosamine, N-
acetylgalactosamine, and additional mannoses, and then fucose or
NANA as terminal groups) to produce a complex glycoprotein, or
they are not processed further, leaving branched, mannose-
containing chains in a high-mannose glycoprotein
• The ultimate fate of N-linked glycoproteins is the same as that of the
O-linked, e.g., they can be released by cell, become part of a CM, or
alternatively, N-linked glycoproteins can be translocated to the
lysosomes
46. 3. Enzymes destined for lysosomes:
• N-linked glycoproteins being processed through Golgi
can be phosphorylated at one or or more specific
mannosyl residues.
• Mannose 6-P receptors, located in Golgi, bind the
mannose 6-P residues of these targeted enz’s, resulting
in their translocation to the lysosomes
• I-cell disease is a rare syndrome in which the acid
hydrolase enz’s normally found in lysosomes are absent,
resulting in an accumulation of substrates normally
degraded by lysosomal enz’s within these vesicles
Note: I-cell disease is so-named because of the large
inclusion bodies seen in cells of patients with this
disease
• In addition, high amounts of lysosomal enz’s are found in
patient’s plasma, suggesting that targeting process to
lysosomes (rather than the synthetic pathway of these
enzymes) is deficient.
47. • It has been determined that individuals with I-cell
disease are lacking the enzymic ability to
phosphorylate the mannose residues of potential
lysosomal enz’s, causing an incorrect targeting
of these proteins to extracellular sites, rather
than lysosomal vesicles
• I-cell disease is characterized by skeletal
abnormalities, restricted joint movement, coarse
facial features, & severe psychomotor impairment.
Death usually occurs by age 8 yrs.
49. IX. Lysosomal degradation of glycoproteins
• Degradation of glycoproteins is similar to that of GAGs.
• The lysosomal hydrolytic enz’s are each generally specific
for the removal of one component of the glycoprotein.
• They are primarily exoenzymes that remove their
respective groups in sequence in the reverse order of their
incorporation (“last on, first off”)
• If any one degradative enz is missing, degradation by the
other exoenzymes can’t continue.
• A group of genetic diseases called the glycoprotein storage
diseases (oligosaccharidoses), caused by a deficiency of
one of the degradative enz’s, results in accumulation of
partially degraded structures in lysosomes.
• After cell death, the oligosacch fragments appear in the
urine
Note: these disorders are very often directly associated with
the same enzyme deficiencies involved in
mucopolysaccharidoses & the inability to degrade
glycolipids
50. Summary
• GAGs are long, negatively charged, unbranched
heteropolysacch chains generally composed of a repeating
disaccharide unit [acidic sugar-amino sugar]n
• The amino sugar is either D-glucoamine or D-galactosamine
in which the amino group is usually acetylated, thus
eliminating its +ve charge
• The amino sugar may also be sulfated on C-4 or 6 or on a
non-acetylated nitrogen.
• The acidic sugar is either D-glucuronic acid or its C-5
epimer, L-iduronic acid. These cpds bind large amounts of
water, thereby producing the gel-like matrix that forms the
basis of the body’s ground substance
• The viscous, lubricating properties of mucous secretions are
also caused by the presence of GAGs which led to the
original naming of these cpds as mucopolysaccharides
• As essential components of cell surfaces, GAGs play an
important role in mediating cell-cell signaling & adhesion.
51. • There are 6 major classes of GAGs, including chondroitin
4- & 6-sulfates, keratan sulfate, dermatan sulfate, heparin,
heparan sulfate & hyaluronic acid.
• All of the GAGs, except hyaluronic acid, are found
covalently attached to protein, forming proteoglycan
monomers, which consist of a core protein to which the
linear GAG chains are covalently attached.
• The proteoglycan monomers associate with a molecule of
hyaluronic acid to form proteoglycan aggregates
• GAGs are synthesized in the ER & Golgi. The polysacch
chains are elongated by sequential addition of alternating
acidic & amino sugars, donated by their UDP-derivatives.
The last step in synthesis is the sulfation of some of the
amino sugars. The source of the sulfate is 3’-
phosphoadenosyl-5`-phosphosulfate.
52. • GAGs are degraded by lysosomal hydrolases. They are 1st
broken down to oligosacch’s, which are degraded
sequentially from the non-reducing end of each chain.
• A deficiency of one of the hydrolases results in a
mucopolysaccharidoses. These are hereditary disorders in
which GAGs accumulate in tissues, causing symptoms such
as skeletal & extracellular matrix deformities, & mental
retardation. Examples of these genetic diseases include
Hunter & Hurler syndromes.
• Glycoproteins are proteins to which oligosacch’s are
covalently attached. They differ from proteoglycans in that
length of glycoprotein’s CHO chain is relatively short
(usually 2-10 sugar residues long, although they can be
longer). The CHO’s of glycoproteins do not have serial
repeats as do GAGs.
• Memb-bound glycoproteins participate in a broad range of
cellular phenomena, including cell surface recognition (by
other cells, hormones, viruses), cell surface antigenicity
(e.g., blood group antigens), & components of the
extracellular matrix & of the mucins of the GI & urogenital
tracts, where they act as protective biologic lubricants.
53. • In addition, almost all of the globular proteins present in human
plasma are glycoproteins
• Glycoproteins are synthesized in the ER & Golgi. The
precursors of the CHO components of glycoproteins are sugar
nucleotides. O-linked glycoproteins are synthesized by the
sequential transfer of sugars from their nucleotide carriers to
the protein.
• N-linked glycoproteins contain varying amounts of mannose.
They are synthesized by the transfer of a pre-formed
oligosacch from its membrane lipid carrier, dolichol, to the
protein.
• They also require dolichol, an intermediate carrier of the
growing oligosacch chain.
• A deficiency in the phosphorylation of mannose residues in N-
linked glycoprotein pre-enzymes destined for the lysosomes
results in I-cell disease.
• Glycoproteins are degraded in lysosomes by acid hydrolases. A
deficiency of one of these enz’s results in a glycoprotein
storage disease (oligosaccharidosis), resulting in accumulation
of partially degraded structures in the lysosome.
