3. Living tissue can be thought of as a dynamic meshwork of cells and liquid.
Despite their close proximity to each other, the cells of a tissue are not
simply tightly wound together.
Instead, they are spaced out with the help of the extracellular meshwork.
The matrix will act as a kind of filler that lies between the otherwise tightly
packed cells in a tissue.
Furthermore, not only is the matrix filling the gaps in between these cells
but it is also retaining a level of water and homeostatic balance
4. Overview
Basic components of ECM:
- structural proteins (e.g.,
collagens)
- proteoglycans (GAGs + protein
backbone)
- adhesion proteins (link
components of matrix to each other
and to cells)
Collagens, elastin and laminin are
the principal structural proteins of
connective tissue
6. Proteoglycans
Form gel of ECM
Found in interstitial connective tissue
- synovial fluid, vitreous humor, arterial walls, bone, cartilage, cornea
variety of proteins in the matrix: collagen, elastin, fibronectin, laminin
7. Proteoglycans
Consist of polysaccharide called glycosaminoglycans (GAGs) linked to a core protein
GAGs are composed of repeating units of disaccharides
Proteoglycan may contain >100 GAG chains and consist of up to 95% carbohydrate by
weight
Interact with variety of proteins in the matrix: collagen, elastin, fibronectin, laminin
8. Glycosaminoglycans
GAGs are long, unbranched, heteropolysaccharide composed of repeating
disaccharide chains where one of the sugars is an N-acetylated amino
sugar, either N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine
(GalNAc) and the other is an acidic sugar.
A single exception is keratan sulfate, which contains galactose rather than
an acidic sugar.
The amino sugar is either D-glucosamine or D-galactosamine, in which the
amino group is usually acetylated, eliminating its positive charge
The acidic sugar is either D-glucuronic acid or its C-5 epimer L-iduronic
acid . These uronic sugars contain carboxyl groups that are negatively
charged at physiologic pH and, together with the sulfate groups (−SO4
2−), give GAGs their strongly negative nature
10. Proteoglycans
Negatively charged carboxylate and sulfate groups on the proteoglycan bind positively charged
ions and form hydrogen bonds with trapped water molecules hydrated gel
The Gel
- provides a flexible mechanical support for ECM
- acts as a filter: allows the diffusion of ions, H20, small molecules
- slows diffusion of proteins and movement of cell
- acts as a lubricant
Hyaluronan – the only GAG occurring as a single long polysaccharide chain; the only GAG – not
sulfated. glycosaminoglycan distributed widely throughout connective, epithelial, and neural
tissues.
11. Because of the high concentration of negative charges, these
repeating disaccharide chains tend to be extended in solution.
They repel each other and are surrounded by a shell of water
molecules
When a solution containing GAGs is compressed, the water is
squeezed out, and 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 the repulsion
of their negative charges. This property contributes to the
resilience of cartilage, synovial fluid, and the vitreous humor of
the eye
12. Proteoglycans - structure
Contain many chains of GAGs (mucopolysaccharides)
Function extracellularly
Long, negatively charged glycosaminoglycan chains repel each other
occupy a very large space and act as “molecular sieves”,
determining which substances enter/leave cells
GAGs differ in the monosaccharides present in their repeating
disaccharide units: chondroitin sulfate, dermatan sulfate, heparin,
heparin sulfate, hyaluronic acid, keratin sulfates I and II
GAGs are linked to proteins (except for hyaluronic acid)
- usually attached covalently to serine or threonine residues
- keratin sulfate I is attached to asparagine
13. GAGs–protein linkage: GAGs attached to
core protein via covalent linkage are most
commonly through a trihexoside
(galactose– galactose–xylose) and a serine
residue in the protein. An Oglycosidic bond
is formed between the xylose and the
hydroxyl group of the serine.
14. Many proteoglycan monomers can associate
with one molecule of hyaluronic acid to form proteoglycan
aggregates. The association is not covalent and occurs primarily
through ionic interactions between the core protein and the
hyaluronic acid. The association is stabilized by additional small
proteins called link proteins
17. Proteoglycans - function
determinate which substances enter/leave cells
Give resilience and flexibility to substances (e.g., cartilage) – permitting compression and reexpansion of molecule
In cartilage proteoglycans, chondroitin sulfate and keratin sulfate are the main types of GAGs.
18. Clinical correlate
ECM not only holds the cells together, but also serves to keep cells from moving to other locations and
prevents large molecules and particles (e.g., microorganisms) from reaching contiguous and distant cells
Infections spread, in part, because the infectious agent alters the “containing” capacity of the ECM
Cancer cells that metastasize can do so only by altering the integrity of matrix
Disease like rheumatoid arthritis and osteoarthritis involve damage to the functional capacity of the matrix
Alterations in the matrix of the renal glomerulus allows proteins to be excreted into urine
Genetic defects structurally and functionally abnormal matrix connective tissue disorders (e.g., Ehlers-
Danlos syndrome, Marfan syndrome)
Deficiencies of lysosomal enzymes involved in normal degradation of matrix molecules
mucopolysaccharidoses
19. Clinical correlate
Functional properties of a normal joint depend, in part, on the presence of a soft, well-lubricated,
deformable, and compressible layer of cartilaginous tissue
- covers the ends of the long bones that constitute joint
20. Proteoglycans - synthesis
Protein component of proteoglycans – synthesized on the ER
Enters the lumen of ER, where the initial glycosylations occur
Proteoglycans are synthesized in the ER and Golgi complex
- Synthesis of the proteoglycans starts with the attachment of a sugar to a serine, threonine, or asparagine
residue of the protein
- Additional sugars, donated by UDP-sugar precursors, add sequentially to the nonreducing end of the
molecule
- Glycosylation occurs initially in the lumen of the ER and subsequently in the Golgi complex
Glycosyltransferases – enzymes that add sugars to the chain
- specific for the sugar being added, the type of linkage that is formed, and the sugars already present in
the chain
Once the initial sugars are attached to the protein, the alternating action of 2 glycosyltransferases adds the
sugars of repeating disaccharide to the GAG chain
Sulfation occurs after addition of sugar
- 3’-phosphoadenosine 5’-phosphosulfate (PAPS) = active sulfate: provides sulfate groups
Epimerase converts glucuronic acid residues to iduronic acid residues
21. Bottlebrush structure of a proteoglycan,
with a magnified segment
• After synthesis, proteoglycan is secreted from the cell
• Final structure resembles a bottlebrush, with many GAG
chains extending from the core protein
22. Proteoglycan aggregate
Many proteoglycan monomers can associate with
one molecule of hyaluronic acid to form
proteoglycan aggregates. The association is not
covalent and occurs primarily through ionic
interactions between the core protein and the
hyaluronic acid. The association is stabilized by
additional small proteins called link proteins
23. Interactions between the cell
membrane and the
components of ECM
The proteoglycans interact with
adhesion protein, fibronectin, which is
attached to the cell membrane protein
integrin
- cross-linked fibers of collagen also
associate with this complex ECM
24. Proteoglycans in cartilage
Long polysaccharide side chains of proteoglycans in cartilage contain many anionic groups
High concentration of negative charges attracts cations that create a high osmotic pressure within
cartilage, drawing water into this specialized connective tissue and placing collagen network under
tension
At equilibrium, tension balances the swelling pressure caused by proteoglycans
The complementary roles of this macromolecular organization give cartilage its resilience
Cartilage can withstand the compressive load of weight bearing and then re-expand to its previous
dimensions when that load is relieved
25. Synthesis of chondroitin
sulfate
Sugars are added to the protein one at a time
- UDP-sugars serve as precursors
A xylose residue is added to a serine in the
protein
Then 2 galactose residues are added,
followed by glucuronic acid (GlcUA) and an
N0acetylglucosamine (GalNac)
Subeqquent additions occur by the
alternating action of 2 enzymes that produce
the repeating disaccharide units
26. Proteoglycans - degradation
Lysosomal enzymes degrade proteoglycans, glycoproteins, and glycolipids
- brought into the cell by endocytosis
Lysosomes fuse with the endocytic vesicles
Lysosomal proteases digest the protein component
Carbohydrate component is degraded by lysosomal glycosidases
- endoglycosidases – cleave chains into shorter oligosaccharides
- exoglycosidases – specific for each type of linkage, removes the sugar residues, one at a time,
from reducing ends
27. Mycopolysaccharidoses
An inability to degrade proteoglycans mucopolysaccharidoses
Deficiencies of lysosomal glycosidases
Deficiencises of lysosomal glycosidases partially degraded carbohydrates from proteoglycans,
glycoproteins, glycolipids accumulate within membrane-enclosed vesicles inside cells
enlargement of organ with impairment of it function
Skeleton deformities, developmental delay, impaired cognitive abilities
28. Mucopolysaccharidoses are hereditary diseases (approximately 1:25,000 live
births) caused by a deficiency of any one of the lysosomal hydrolases normally
involved in the degradation of heparan sulfate, dermatan sulfate, and/or
keratin sulfate They are progressive disorders characterized by lysosomal
accumulation of GAGs in various tissues, causing a range of symptoms, such as
skeletal and ECM deformities, and intellectual disability. All are autosomal-
recessive disorders except Hunter syndrome, which has X-linked inheritance. .
Diagnosis is confirmed by measuring the patient’s cellular level of the
lysosomal hydrolases. Bone marrow and cord blood transplants, in which
transplanted macrophages produce the enzymes that degrade GAGs, have
been used to treat Hurler and Hunter syndromes, with limited success. Enzyme
replacement therapy is available for both syndromes but does not prevent
neurologic damage
31. I-Cell Disease
I-Cell disease is a rare lysosomal storage disease named for large inclusion
bodies seen in cells of patients with the disease. GlcNAc
phosphotransferase is deficient and mannose 6-phosophate is not
generated on proteins destined for lysosomes.
Lack of M6P on amino acid residues causes precursor acid hydrolases to
traffic to the plasma membrane and be secreted constitutively, instead of
trafficking to lysosomes.
Consequently, the acid hydrolases are absent from lysosomes, and the
macromolecule substrates for these digestive enzymes accumulate within
the lysosomes, generating the inclusion bodies that define the disorder.
32. In addition, high concentrations of lysosomal enzymes are found in the
patient’s plasma and urine, indicating that the targeting process to
lysosomes is deficient.
I-Cell disease is characterized by skeletal abnormalities, restricted joint
movement, coarse (dysmorphic) facial features, and severe psychomotor
impairment. Because I-cell disease has features in common with the
mucopolysaccharidoses and sphingolipidoses, it is termed a mucolipidosis
(ML II).
Currently, there is no cure, and death from cardiopulmonary complications
usually occurs in early childhood. Pseudo-Hurler polydystrophy (ML III) is a
less severe mucolipidosis form of I-cell disease, in which the
phosphotransferase maintains some residual enzymatic activity, and it
symptomatically resembles a mild form of Hurler syndrome.
33.
34. integrins
Integrins – major cellular receptors for ECM proteins
Provide a link between the internal cytoskeleton of cells (primarily actin) and extracellular proteins (eg, fibronectin,
collagen, laminin
Involved in signaling options
Consist of alpha and beta subunit
24 unique alpha/beta dimers have been discovered
Can be activated by “inside-out” or “outside-in” mechanisms
Certain integrins, such as those associated WBCs, are normally inactive, and get activated during infection
- activation allows them to bind to vascular endothelial cells (leukocyte adhesion) at the site of infection
Leukocyte adhesion deficiency (LAD) is a genetic disorder that results from mutations in the beta2 integrin
- leukocytes can not be recruited to the sites of infection
drugs have been developed to block either beta2 or alpha 4 integrins (on lymphocytes) to treat inflammatory and
autoimmune disorders by interfering with the normal WBC response to cytokines
35. Adhesion proteins
Members: fibronectin, laminin
Extracellular glycoproteins
Link integrins to ECM components
Contain separate distinct binding domains for proteoglycans, collagen , and fibrin
- allow to bind the various components of the ECM
Contain specific binding domains – for cell surface receptors – integrins
- integrins bind to fibronectin on the external surface, span the plasma membrane and
adhere to proteins, which, in turn, bind to the intracellular actin filaments of the
cytoskeleton
- also provide a mechanism for signaling between cells via both internal and external
signals
36. Key concepts
Extracellular matrix IECM) consists of fibrous structural proteins, proteoglycans, and adhesion proteins
ECM provides support to the tissues and restricts movement of cells
Proteoglycans consist of polysaccharides (glycosaminoglycans) bound to a core protein
- polysaccharides are usually a repeating disaccharide unit, containing negative charges
- because of charge repulsion, the proteoglycans form a hydrated gel that provides flexible
mechanical support to the ECM
Integrins are cellular membrane receptors for ECM proteins and they link the cellular cytoskeleton to
extracellular protein
Integrins are also signaling proteins when they are bound to appropriate compounds
Adhesion proteins link the integrins to ECM components
Matrix metalloproteinasesare the only proteases that can degrade ECM components, and they are
carefully regulated by the tissue inhibitors of matrix metalloproteinases (TIMPS)
