Transcript Hyaluronan and Proteoglycans

Proteoglycans and
Glycosaminoglycans
Natasha E. Zachara Ph.D.
[email protected]
The Department of Biological Chemistry
Key to Glycan Structures
Both editions of Essentials of Glycobiology are freely available online.
Focus on Chapters 15, 16, 35, 37 and 44.
Other articles used in the preparation of this lecture are listed at the bottom of the
relevant slide.
Chapter 1, Figure 5
Essentials of Glycobiology
Second Edition
Classes of Glycoproteins:
•
O-linked glycosylation, describes carbohydrates bound to the
protein backbone through hydroxyl residues, such as those in serine
(Ser), threonine (Thr), tyrosine (Tyr), hydroxylysine (hLys), and
hydroxyproline (hPro).
– Mucin-like
– Phosphoglycosylation
– O-GlcNAc and
• N-linked glycosylation, typically refers to the amide bond
formed between GlcNAc and Asn in the b-conformation.
•
C-mannosylation, is the attachment of an a-Man to carbon-2 of the
indole ring of tryptophan (Trp).
•
Glycosylphosphatidyl inositol (GPI)-anchors, GPI-anchors link the
terminal residue of a protein through phosphoethanolamine and a
carbohydrate core to a lipid moiety which anchors the protein in the
lipid bilayer.
•
Proteoglycans: Proteins that are glycosylated by one or more
glycosaminoglycan chains.
What are:
• Proteoglycans: Any protein with one or more covalently attached
glycosaminoglycan chains.
• Glycosaminoglycans (GAGs): Polysaccharide side chains of
proteoglycans or free complex polysaccharides composed of
linear disaccharide repeating units, each composed of a
hexosamine and a hexose or a hexuronic acid.
Glycosaminoglycans include: Heparin, Heparan sulfate,
Chondroitin sulfate, Dermatan sulfate, and Hyaluronan.
• Hyaluronan or hyaluronic acid: A glycosaminoglycan defined by
the disaccharide unit (GlcNAcβ1–4GlcAβ 1–3) n that is neither
sulfated nor covalently linked to protein. Hyaluronan is NOT
typically added to proteins, which is unlike other
glycosaminoglycans.
Introduction: Proteoglycans
• Also known as mucopolysaccharides:
– First studies proteoglycans included an
anticoagulant (heparin) from liver and
chondromucoid from the cartilage
• The glycosaminoglycan chains of
proteoglycans are linear and can be up to 80
sugars long:
– >1 GAG can be linked to a protein (for example
aggrecan, >100 GAGs)
– These can form gels, similar to mucins
• Present in virtually all extracellular matrices:
– Interact with fibrous proteins that provide tensile
strength and elasticity, as well as and adhesive
glycoproteins to provide a hydrated gel that
resists compressive forces
Cartilage – proteoglycan
From: http://www.davidlnelson.md/Osteoarthritis.htm
http://en.wikipedia.org/wiki/Heparin
Proteoglycans can be membrane bound
or secreted
Chapter 16, Figure 2
Essentials of Glycobiology
Second Edition
Proteoglycan Components
• Proteoglycans display structural variation:
There are a large number of core proteins (20-450kDa)
These can be modified by one or more types of glycosaminoglycan
There is variation in chain length, and stoichiometry
Proteoglycans can also be modified by O-linked and N-linked glycans, as
well as GPI anchors
– The GAG component can be further modified by sulfation,
phosphorylation, fucosylation, and sialylation
–
–
–
–
http://www.msdlatinamerica.com/ebooks/PracticalOrthopaedicSportsMedicineArthrocopy/sid360574.html
Who are the protein components?
CS: Chondroitin sulfate;
DS: Dermatan sulfate;
GPI: Glycosylphosphatidylinositol;
HS: Heparan sulfate;
KS: Keratan sulfate;
SLRP: Small leucine rich family of
proteoglycans.
Couchman J R , and Pataki C A
J Histochem Cytochem 2012;60:885-897
Copyright © by The Histochemical Society
GAG can be Classed into 6 Major Types, of Which
Five are Found Covalently Bound to Proteins
①
②
③
④
⑤
⑥
Heparin
Heparan sulfate
Chondroitin sulfate
Dermatan sulfate
Keratan sulfate
Hyaluronan (not typically added to proteins)
What do these all have in common:
➤
Repeating units of hexosamine and a hexose or a hexuronic acid
n
n
Glycosaminoglycans consist of
repeating disaccharide units
GlcNAc
GalNAc
Galactose
Glucuronic acid
Iduronic acid
Chapter 16, Figure 3
Second Edition
Essentials of Glycobiology
??? 6S, 4S, 2S, NS ???
http://westpalmbeach.injuryboard.com/defective-and-dangerous-products/heparin-or-over-sulfatedchondroitin-sulfate-that-is-the-question.aspx?googleid=251088
Heparin versus Heparan Sulfate
HS is made by virtually all cell types, whereas Heparin is produced in Mast cells
Chapter 16, Table 7
Essentials of Glycobiology
Biosynthesis of Proteoglycans
Chapter 3 Figure 1
Essentials of Glycobiology
Keratan Sulfate
• KS consists of a polysulfated poly-Nacetyllactosamine structure identical
to that found on conventional
glycoproteins and mucins
•
Keratan Sulfate I: N-glycan core
– ~50 disaccharides, 20–25 kD
– a mixture of non-sulfated, monosulfated (Gal-GlcNAc6S), and disulfated (Gal6S-GlcNAc6S)
disaccharide units.
– Sulfation of a terminal galactose
blocks chain elongation
•
Keratan Sulfate II: O-glycan care
(Ser/Thr)
Chapter 16, Figure 4
Essentials of Glycobiology
Second Edition
The Synthesis of Chondroitin Sulfate, Dermatan Sulfate, Heparin
and Heparan Sulfate is Initiated by the Same Enzyme in the ER
GlcNAc
GalNAc
Galactose
Glucuronic acid
Iduronic acid
Chapter 16, Figure 3
Second Edition
Essentials of Glycobiology
The biosynthesis of CS and HS is initiated by the
formation of a linkage region tetrasaccharide
A glycine residue is often
found on the carboxy
terminal side of the serine
attachment site. 2 Acidic
residues nearby the
glycosylated serine are
also common features.
But, there is no strict
consensus motif..
These enzymes are important
control points because they
ultimately regulate the type
of glycosaminoglycan chain
assembled. There is some
evidence that the
surrounding amino acid
dictates recognition.
Chapter 16, Figure 5
Essentials of Glycobiology
Second Edition
Biosynthesis of chondroitin sulfate/dermatan sulfate
Chapter 16, Figure 6
Essentials of Glycobiology
Second Edition
Biosynthesis of heparan sulfate
Extl3 recognizes a
linear acidic sequence
in proteins.
Chapter 16, Figure 7
Essentials of Glycobiology
Second Edition
Sulfation provides a finger print which define
different biological functions
In contrast to chondroitin chains, which tend to have long tracts of fully
modified disaccharides, the modification reactions in HS biosynthesis occur
in clusters along the chain, with regions devoid of sulfate separating the
modified tracts. This leads to NA domains, NA/NS domains, and NS domains.
Essentials of Glycobiology
Second Edition
Chapter 16, Figure 8
Functions of Proteoglycans
• Interstitial Proteoglycans:
– Bind water and form hydrated matrices
– Fills the space between cells
– Can absorb compressive loads
• Help organize the basement membranes, providing a scaffold
for epithelial cell migration, proliferation, and differentiation
• Can regulate the permeability of specialized BM.
• Can bind cytokines, chemokines and protect them from
proteolysis, facilitating the formation of morphogen gradients
• Can act as co-receptors for tyrosine kinases.
Interstitial Proteoglycans: Aggrecan
•
Aggrecan family:
– Aggrecan family of proteoglycans consists of aggrecan, versican,
brevican, and neurocan
– The interstitial proteoglycans appear to be unique to vertebrates
– Characterized by a N-terminal Hyaluronan binding domain
– C-terminal lectin domain
– Generally modified by Chondroitin sulfate, and occasionally keratan
sulfate
http://www.sigmaaldrich.com/catalog/product/sigma/a1960?lang=en&region=US
Aggrecan
Aggrecan is a critical component for
cartilage structure and the function of
joints;
Can be modified by >100 GAG chains.
Forms a gel with the ability to resist
compressive loads
Functionally, the G1 domain interacts
with hyaluronan and link protein, forming
stable ternary complexes in the
extracellular matrix
Aggrecan plays an important role in
mediating chondrocyte-chondrocyte
and chondrocyte-matrix interactions
through its ability to bind hyaluronan
http://www.academicwebpages.com/preview/other/grodzinsky/research/aggrecan.html
Interstitial Proteoglycans: SLRPs
• Small leucine rich proteoglycans (SLRPs)
– 9 members
– Modified by Dermatan sulfate, chondroitin
sulfate, keratan sulfate
– Interacts with collagen
• Stabilizes collagen in tendons
– In the eye:
• Decorin, Lumican, Keratocan, Mimecan
• Relatively uniform size with only 1-2 Gags
• maintain the even spacing of type I collagen
fibrils in the cornea
• Fit between collagen fibers in the eye, maintain
the spacing and thus transparency
• Mutations in Keratan sulfate synthesis lead to
macular corneal dystrophy
Membrane Bound Proteoglycans
• For example Syndecan
– Single membrane pass
• Facilitate cellular
interactions
• Binding of Syndecan to HA
can induce
oligomerization; and
recruitment of Kinases,
PDZ-domain proteins and
cytoskeletal components
to the cytoplasmic
domain
• Glypican
– Characterized by GPIAnchors
– An N-terminal globular
domain
– Only carry HS
– Bind numerous factors
essential for morphogenesis
and development
• GPC3: loss of GPC3 results in
Simpson-Golabi-Behmel
syndrome, characterized as
an overgrowth disorder –
suggesting that GPC3 acts
to inhibit cell growth.
Binding of growth factors to proteoglycans
can induce oligomerization and thus signaling
Trends in Cell Biology, Volume 19, Issue 3, 2009, 119 - 129
Many functions of proteoglycans are
mediated by proteins which bind GAGs
Class
Example
Physiological/pathophysiologic
al effect
Enzymes
glycosaminoglycan biosynthetic enzymes, thrombin and coagulation factors
(proteases), complement proteins (esterases), extracellular superoxide
dismutase, topoisomerase
multiple
Enzyme inhibitors
antithrombin III, heparin cofactor II, secretory leukocyte proteinase inhibitor,
C1-esterase inhibitor
coagulation, inflammation,
complement regulation
Cell adhesion
proteins
P-selectin, L-selectin, some integrins
cell adhesion, inflammation,
metastasis
Extracellular
matrix proteins
laminin, fibronectin, collagens, thrombospondin, vitronectin, tenascin
cell adhesion, matrix
organization
Chemokines
platelet factor IV, γ-interferon, interleukins
chemotaxis, signaling,
inflammation
Growth factors
fibroblast growth factors, hepatocyte growth factor, vascular endothelial
growth factor, insulin-like growth factor–binding proteins, TGF-β-binding
proteins
mitogenesis, cell migration
Morphogens
hedgehogs, TGF-β family members
cell specification, tissue
differentiation, development
Tyrosine-kinase
growth factor
receptors
fibroblast growth factor receptors, vascular endothelium growth factor
receptor
mitogenesis
Lipid-binding
proteins
apolipoproteins E and B, lipoprotein lipase, hepatic lipase, annexins
lipid metabolism, cell membrane
functions
Adaptation, Table 35.1 Essentials in Glycobiology
Table 35.3 Examples of oligosaccharides preferentially recognized by
glycosaminoglycan-binding proteins
Protein
Glycosaminoglycan
partner
Antithrombin
heparin/heparan sulfate
Fibroblast growth factor 2
heparin/heparan sulfate
Lipoproteinlipase
heparin/heparan sulfate
Heparin cofactor II
dermatan sulfate
Herpes simplex virus
Glycoprotein gD
heparin/heparan sulfate
Oligosaccharide
Essentials of Glycobiology
Chapter 35, Table 35.3
Proteoglycans and Signaling Gradients
https://www.neb.com/sitecore/content/nebsg/home/applications/glycobiology/depolymerization-of-heparin-hs
The FGF Receptor & Heparin
• >22 growth factors bind heparin
• FGF2 has a very high affinity for
heparin (Kd ~ 10−9 M)
• FGF2 has potent mitogenic
activity in cells
• Heparin promotes the mitogenic
response by promoting
dimerization of the FGF receptor
• Technically a pentasaccharide is
required for binding, although it’s
only longer oligomers that trigger
a biological response
http://9e.devbio.com/article.php?ch=3&id=61
Congenital Exostosis
•
Defects in the formation of heparan sulfate (HS) cause hereditary
multiple exostosis (HME)
•
an autosomal dominant disease with a prevalence of about 1:50,000
•
It is caused by mutations in two genes EXT1 and EXT2, which are
involved in HS synthesis.
•
HME patients have bony outgrowths, usually at the growth plates of
the long bones.
•
HME mutations occur in EXT1 (60–70%) and EXT2 (30–40%). However,
the partial loss of one allele of either gene appears sufficient to cause
HME. This means that haplo-insufficiency decreases the amount of HS
and that EXT activity is rate limiting for HS biosynthesis.
•
The mechanism of HME pathology is likely rooted in a disruption of the
normal distribution of HS-binding growth factors, which include FGF
and morphogens such as hedgehog, Wnt, and members of the TGF-β
family.
How does disrupting signaling alter neuronal
development?
•
Heparan sulfate was eliminated from
postnatal neurons by conditionally
inactivating Ext1, the gene encoding an
enzyme essential for heparan sulfate
synthesis.
•
Mutant mice recapitulated a range of
autistic symptoms, including impairments in
social interaction, expression of stereotyped,
repetitive behavior, and impairments in
ultrasonic vocalization.
•
From these and other experiments they
concluded that AMPA receptor-mediated
synaptic transmission is compromised in the
absence of HS, presumably because of the
reduced synaptic expression of AMPA
receptors.
Proc Natl Acad Sci U S A. 2012 Mar 27;109(13):5052-6. doi:
10.1073/pnas.1117881109. Epub 2012 Mar 12. PMID: 2241180
Proteoglycans and Signaling Gradients
• Mutations in the Heparin
Sulfate biosynthetic
machinery lead to
defects in Wnt signaling.
Cold Spring Harb Perspect Biol. 2009 Sep;1(3):a002493. PMID: 20066107
Proteoglycans and Signaling Gradients
• Mutations in the Heparin
Sulfate biosynthetic
machinery lead to
defects in Wnt signaling;
• Wnt can not diffuse
across cells without two
glypicans, Dally and Dlp.
• There is also evidence
suggesting that Dally
presents Wnt to the dFz2
signaling receptor,
promoting an activation
of signaling.
Bornemann D J et al. Development 2004;131:1927-1938
Cells Secrete Heparanase to Release
Growth Factors
Extracellular heparanase can
cleave HS chains at restricted sites,
resulting in release of growth
factors or chemokines immobilized
on HS proteoglycans at cell
surfaces or in the ECM.
The sulfation of Heparan Sulfate
can also be modulated at the cell
surface, resulting in an altered
response of cells to growth factors
and morphogens.
Essentials of Glycobiology
Second Edition
Chapter 16, Figure 10
Anti-Thrombin & Heparin/HS Interactions
• heparin is used clinically as an anticoagulant
http://www.neurology.org/content/78/7/501F2.expansion.html
Anti-Thrombin & Heparin/HS Interactions
• heparin is used clinically as an
anticoagulant
• Anti-thrombin is a protease
inhibitor of the Serpin family
• Heparin activates anti-thrombin
– Change in conformation which
results in a 1000x enhancement
of the rate of inactivation of
thrombin and factor Xa
– Heparin also acts to bring
thrombin and anti-thrombin
together
Chapter 35, Figure 2
Essentials of Glycobiology
Second Edition
Degradation of GAGs
Essentials of Glycobiology
Second Edition
Chapter 16, Figure 9
Mucopolysaccharidosis
• Defects in GAG catabolism
• A group of rare inherited lysosomal storage disorders
– Characterized by:
• abnormalities in multiple organs
• Reduced life expectancy
• Heterogeneous and progressive
– MPS I, II, and IV can be treated with enzyme replacement
therapy
Muenzer (2011), Rheumatology, 50: V4-V12
MPS 1
•
There are three major types of MPS 1, which differ in severity
•
Caused by defects in a-L-iduronidase – which affects the
degradation of dermatan sulfate and Heparan sulfate
– Hurler syndrome – described in 1919
– Scheie syndrome – described in 1962
– Hurker-Scheie – an intermediate phenotype - was discovered later
Chapter 41, Figures 3 & 4
Essentials of Glycobiology
Second Edition
The Genetic Defect
•
Autosomal recessively inherited
•
There are more than 100 different alleles of the a-L-iduronidase
gene that cause all three forms of MPS I;
– Thus, the designation of MPS type is arbitrary
– In the mildest form, patients may remain undiagnosed for years
– 653 amino acid protein, encoded from a gene on 4p16.3
•
MPS1 mutations include:
– Introduction of premature stop codons (W402X, Q70X)
• These are the most common mutations (70%),
• >13 additional mutations that introduce stop codons
• No detectable protein
–
D349N, E182A and E299A
• Lead to reduced protein levels
• Alter protein folding, leading to export and degradation from the ER
– A75T does not alter protein levels, but ablates enzyme activity
Treatments
➤
Enzyme replacement therapy (ERT)
➤
➤
➤
➤
➤
Laronidase (Genzyme), approved since 2003
Infusions every other week (1-4h)
Clinical outcomes: Reduced urinary GAGs, reduced liver volume, improved
vital capacity, improved walking tests
Risks: anaphylaxis
Haematopoitic stem cell transplantation (HSCT)
➤
➤
➤
➤
Bone marrow transplantation most effective in young patients
Best treatment for more severe MPS 1
Improves median age from 6.8 years to beyond 20 years
Maintains cognition, but musculoskeletal disease continues to progress along
with reduced vision and poor growth.
➤
These are not ideal, in part as secondary damage from GAG
accumulation is often irreversible;
➤
Early diagnosis improves treatment outcomes.
What are:
• Proteoglycans: Any protein with one or more covalently
attached glycosaminoglycan chains.
• Glycosaminoglycans: Polysaccharide side chains of
proteoglycans or free complex polysaccharides composed of
linear disaccharide repeating units, each composed of a
hexosamine and a hexose or a hexuronic acid.
Glycosaminoglycans include: Heparin, Heparan sulfate,
Chondroitin sulfate, Dermatan sulfate, and Hyaluronan.
• Hyaluronan or hyaluronic acid: A glycosaminoglycan defined
by the disaccharide unit (GlcNAcβ1–4GlcAβ 1–3) n that is
neither sulfated nor covalently linked to protein.
What is Hyaluronan?
•
A high molecular weight
glycosaminoglycan;
•
Purified from bovine vitreous
humor in the 1930’s by Myer and
Palmer (1934);
•
hyaluronic acid: from hyaloid
[meaning vitreous] and uronic
acid (as it contained uronic acid
and an amino sugar);
•
HA is not attached to proteins, is
not sulfated or fucosylated, and
contains no Iduronic acid;
•
Largest polysaccharide in
vertebrates.
In the beta configuration of the bulky
groups (hydroxyls, carboxylate moieties,
anomeric carbon) are in the sterically
favorable equatorial positions. Thus, the
structure of the HA disaccharide is
energetically favorable.
Chapter 15, Figure 1
Essentials of Glycobiology
Second Edition
HA arose in evolution at the same time
as the notochord
•
Made by virtually all cells from
vertebrates, and its expression
correlates with tissue
expansion and cell motility;
•
Made by animal cells, but not
Drosophila melanogaster and
Caenorhabditis elegans;
•
Sometimes found in the
capsule of some strains of
Streptococci. This is quite likely
pirated enzymatic machinery
(from vertebrate hosts).
http://www.mhhe.com/biosci/genbio/maderbiology7/graphics/mader07b/online_vrl/images/0619l.jpg
http://www2.drury.edu/educ200/ghyde10-website/Appendicularskeleton.htm
Hyaluronan has a large hydrodynamic volume
•
Can be up to 104 disaccharides long, ~4 ×
106 Daltons;
•
Up to10mM in length, thus stretching half way
around the cell;
•
Polyanionic;
•
At 3-5mg/ml HA occupies all of the solvent;
•
Thus, it can filter out large molecules while
allowing small molecules to pass;
•
HA is stiffened by a combination of the
chemical structure of the disaccharide,
internal hydrogen bonds, and interactions
with solvent; a hyaluronan molecule assumes
an expanded random coil structure in
physiological solutions;
•
Has swelling pressure and high viscosity, and
is essential for distributing load in the joints.
http://www.glycoforum.gr.jp/science/hyaluronan/HA01/HA01E.ht
ml
Hyaluronan has a large hydrodynamic volume
• In some tissues HA can be a major
constituent:
– in the vitreous of the human eye HA
is 0.1-0.4 mg/g wet weight);
– in synovial joint fluid (3-4 mg/ml).
•
The largest amount of hyaluronan
resides in the skin
– (7-8 g per average adult human;
– ~50% of the total in the body.
https://askanesthetician.files.wordpress.com/2011/01/hyaluronic-acid1.jpg
http://www.laurenscharff.com/research/donia/aging_visual_changes.htm
Hyaluronan Biosynthesis
•
HAS or Hyaluronan synthetase is the
bifunctional glycosyltransferase;
•
In mammals there are three
homologs:
– HAS2 appears to be the most
essential;
– Unlike other forms of protein
glycosylation, extension occurs
at what might be considered the
reducing terminal end;
•
HA is exuded into the ECM as part of
the biosynthetic process;
•
5-6 possible trans-membrane
domains;
•
Metabolic studies have shown that
the half life of a hyaluronan molecule
in cartilage is normally 2-3 weeks.
Chapter 15, Figure 2
Essentials of Glycobiology
Second Edition
HA Degradation
•
~5 grams of HA is turned over each
day, ~1/3 of that in the human
body;
•
The endothelial cells of the lymph
nodes and liver sinusoids remove
HA via specific receptors
–
LYVE-1 (a homolog of CD44)
–
HARE (hyaluronan receptor for
endocytosis);
•
Large molecules of HA are clipped
by a GPI-anchored hyaluronidase,
most likely Hyal2;
•
HA is ultimately degraded in the
lysosome.
Chapter 16, Figure 9
Essentials of Glycobiology
Second Edition
HA Function
•
•
•
•
•
HA can play a structural role: it has swelling pressure and high
viscosity, and is essential for distributing load in the joints.
HA increases levels of tissue hydration, which can facilitate movement
of cells through tissues.
Promotes the assembly of of extracellular matrices through specific
interactions with other macromolecules;
HA interacts with several types of cell-surface receptors, especially
CD44 and RHAMM.
Thus, HA plays roles in development, tissue organization, cell
proliferation, diabetes, stress and inflammation;
– HA binding proteins;
– ECM Assembly;
– Ovulation;
– Cell Signaling;
– Fertilization.
Many of Hyaluronan’s Functions are
Dependent on HA binding Proteins
Link domains are
characterized are ~100
amino acids in length
and are similar to C-type
lectins.
These interactions are
calcium independent.
There are alternative
domains, many of which
have a or a BX7B motif.
Chapter 15, Figure 3
Essentials of Glycobiology
Second Edition
Link Proteins: Linking Proteoglycans to Hyaluronan
Hyaluronan often forms a
scaffold for the binding of
other proteoglycans, such
as the connective tissues
surrounding smooth muscle
cells in the aorta and
fibroblasts in the dermis of
skin;
Aggrecan is one protein
with a LINK domain – and
deletion of the Link domain
results in mice with defects
in cartilage and bone
formation.
Chapter 16, Figure 1
Essentials of Glycobiology
Second Edition
In some situations HA can be covalently
linked to a protein: Inter-aTrypsin-inhibitor
•
Inter-αTrypsin Inhibitor (IαI) is found in
serum at 0.5mg/ml;
•
IαI is a trypsin inhibitor, but a poor
inhibitor of physiologically relevant
proteases such as elastase and
kallikrein;
•
Contains two heavy chains linked to
chondroitin sulfate through an ester
linkage to GlcNAc. The chondroitin
sulfate in turn modifies bikunin.
•
The Inter-αTrypsin inhibitor can be
transferred from Chondroitin sulfate to
HA generating the Serum-derived
Hyaluronan-Associated Protein (SHAP)
http://glycoforum.gr.jp/science/hyaluronan/HA22/HA22E.html
Hyaluronan and Ovulation
•
The oocyte is surrounded by closely
adherant cumulus cells to form the
compact COC or cumulus cell-oocyte
complex;
•
Gonadotropin:
– Resumption of meiosis by the oocyte;
– Permeabilization of the follicle to large
serum proteins including the IaI, which is
essential for expansion of the COC;
– Synthesis of a HA (upregulation of has2)
rich ECM, which in part promotes
expansion of the COC. HA reaches a
concentration of ~0.5 mg/ml);
– Crosslinking of the HA, probably via IaI is
essential for expansion;
– HA promotes detachment of the oocytes
from the follicular wall;
– HA also appears to promote the capture
of the release oocyte by the oviduct.
HA, Cell Adhesion, and Locomotion
HA is expressed abundantly during
morphogenesis and in both
physiological and pathological
invasive processes;
Has2-null mouse is embryonic lethal
phenotype;
Has2-null embryonic heart do not
synthesize hyaluronan or undergo
endothelial-mesenchymal
transformation and migration;
Co-culturing Has2 wild-type and null
embryonic cells, or adding
hyaluronan to the culture rescues
the phenotype.
Co-culture
Conditioned media
Hyaluronan
Boiled Hyaluronan
Exp Clin Cardiol. 2001 Spring;6(1):4-10. PMID: 20428437
CD44, a HA binding protein, has altered
expression in many tumors
•
CD44 is a transmembrane receptor expressed
by many cell types. CD44 is heavily
glycosylated and can be subject to
differential mRNA splicing.
•
Binds to hyaluronan, and the interaction can
mediate leukocyte rolling and extravasation;
•
Changes in CD44 expression are associated
with a wide variety of tumors and the
metastatic spread of cancer;
•
When hyaluronan binds to CD44, this
promotes clustering of CD44, and the
cytoplasmic tail interacts with regulatory and
adaptor molecules, such as SRC kinases, RHO
GTPases, VAV2, GAB1, and ankyrin and ezrin.
•
Hyaluronan binding to RHAMM also
transduces signals that influence growth and
motility; SRC, FAK, ERK, and PKC.
Chapter 37, Figure 6
Essentials of Glycobiology
Second Edition
Hyaluronan Oligomers May Antagonize CD44
Based Survival Signaling
• Treatment with short oligomers of
HA sensitized tumor cells to
chemotherapeutic drugs, and
inhibited survival signaling.
J Biol Chem. 2003 Jul 11;278(28):25285-8. Epub 2003 May PMID: 12738783
Conclusions
• Proteoglycans are proteins modified by glycosaminoglycan
chains;
• HA is a GAG which is NOT typically attached to proteins;
• GAGs play essential roles in numerous cellular processes,
which is in part mediated though their interactions with other
proteins;
• Underlying the importance of HA and proteoglycans,
mutations in either the synthetic machinery or the breakdown
machinery have profound effects in mammalian models.