ECM notes

Maria Christina Tabone | Diandra Mifsud

Lecture 12: Extracellular Matrix

From Lippincots:

The extracellular matrix (ECM) is the extracellular part of animal tissue that usually provides

structural support to the animal cells in addition to performing various other important

functions. Due to its diverse nature and composition, the ECM can serve many functions, such

as providing support, segregating tissues from one another, and regulating intercellular

communication. The ECM regulates a cell's dynamic behavior. In addition, it sequesters a wide

range of cellular growth factors, and acts as a local depot for them.

Components of the extracellular matrix:

Collagen forms 90 % of the total weight of bone matrix proteins. It consists mainly of collagen

type I, although trace amounts of other types, such as collagen III, V, XI and XIII have also been

found. Elastin is also found in the extracellular matrix. Non-collagenous proteins are often

thought to be a minor component, because they constrain only ten percent of the bone protein

mass. On the structural basis, four main groups of NCPs are found:

1.) proteoglycans, 2.) g-carboxylated (gla) -proteins, 3.) Glycoproteins and 4.) Others,

including e.g. proteins affecting growth.

GLYCOSAMINOGLYCANS

Glycosaminoglycans are large complexes of negatively charged hetero-polysaccharide chains.

They are generally associated with a small amount of protein, forming proteoglycans, which

typically consist of over 95% carbohydrate. This is in comparison to the glycoproteins, which

consist primarily of protein with a small amount of carbohydrate. Glycosaminoglycans have the

special ability to bind large amounts of water, thereby producing the gel-like matrix that forms

the basis of the bodys ground substance, which, along with fibrous structural proteins such as

collagen and elastin, and adhesive proteins such as fibronectin, make up the extracellular

matrix (ECM). The hydrated glycosaminoglycans serve as a flexible support for the ECM,

interacting with the structural and adhesive proteins, and as a molecular sieve, influencing

movement of materials through the ECM. The viscous, lubricating properties of mucous

secretions also result from the presence of glycosaminoglycans, which led to the original

naming of these compounds as mucopolysaccharides.


Structure of glycosaminoglycans.

Glycosaminoglycans (GAGs) are long, unbranched, hetero polysaccharide chains generally

composed of a repeating disaccharide unit [acidic sugaramino sugar]n. The amino sugar is

either D-glucosamine or D-galactosamine, in which the amino group is usually acetylated, thus

eliminating its positive charge. The amino sugar may

also be sulfated on carbon 4 or 6 or on a

nonacetylated nitrogen. The acidic sugar is either D-

glucuronic acid or its C-5 epimer, L-iduronic acid. 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 and, together with the

sulfate groups, give GAGs their strongly negative

nature.

A. Relationship between glycosaminoglycan structure and function

Because of their large number of negative charges, these heteropolysaccharide chains tend to

be extended in solution. 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 the slippery consistency of mucous

secretions and synovial fluid. When a solution of glycosaminoglycans is compressed, the water

issqueezed out and the glycosaminoglycans are forced to occupy a smaller volume. When the

compression is released, the glycosaminoglycans spring back to their original, hydrated volume


because of the repulsion of their negative charges. This property contributes to the resilience of

synovial fluid and the vitreous humor of the eye.

B. Classification of the glycosaminoglycans : The six major classes of glycosaminoglycans are

divided according to monomeric composition, type of glycosidic linkages, and degree and

location of sulfate units.


C. Structure of proteoglycans

All of the glycosaminoglycans, except hyaluronic acid, are found covalently attached to protein,

forming proteoglycan monomers.

1. Structure of proteoglycan monomers:

A proteoglycan monomer found in cartilage consists of a

core protein to which the linear glycosaminoglycan chains

are covalently attached. These chains, which may each be

composed of more than 100 monosaccharides, extend out

from the core protein, and remain separated from each

other because of charge repulsion. The resulting structure

resembles a bottle brush.

In cartilage proteoglycan, the species of glycosaminoglycans

include chondroitin sulfate and keratan sulfate. Proteoglycans are now grouped into gene

families that code for core proteins with common structural features. The aggrecan family

(aggrecan, versecan, neurocan, and brevican), abundant in cartilage, is an example.

2. Linkage between the carbohydrate chain and the protein:

This linkage is most commonly through a trihexoside (galactose-

galactose-xylose) and a serine residue, respectively. An O-

glycosidic bond is formed between the xylose and the hydroxyl

group of the serine.

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 between

the core protein and the hyaluronic acid. The association is stabilized by additional small proteins

called link

proteins.


III. SYNTHESIS OF GLYCOSAMINOGLYCANS

The polysaccharide chains are elongated by the sequential addition of alternating acidic and

amino sugars donated by their UDP-derivatives. The reactions are catalyzed by a family of

specific glycosyl transferases. The synthesis of the glycosaminoglycans is analogous to that of

glycogen except that the glycosaminoglycans are produced for export from the cell. Their

synthesis occurs, therefore, primarily in the Golgi, rather than in the cytosol.

A. Synthesis of amino sugars

Amino sugars are essential components of glycosaminoglycans, glycoproteins, glycolipids, and

certain oligosaccharides, and are also found in some antibiotics. The synthetic pathway of

amino sugars is very active in connective tissues, where as much as 20% of glucose flows

through this pathway.

1. N-Acetylglucosamine (GlcNAc) and N-acetylgalactosamine(GalNAc):

The monosaccharide fructose 6-phosphate is the pre-cursor of GlcNAc, GalNAc, and the sialic

acids, including N-acetyl neuraminic acid (NANA, a nine-carbon, acidic monosaccharide). In each

of these sugars, a hydroxyl group of the precursor is replaced by an amino group donated by

glutamine. [The amino groups are then almost always acetylated.] The UDP-derivatives of

GlcNAc and GalNAc are synthesized by reactions analogous to those described for UDP-glucose

synthesis. These nucleotide sugars are the activated forms of the monosaccharides that can be

used to elongate the carbohydrate chains.


2. N-Acetylneuraminic acid:

N-Acetylneuraminic acid (NANA) is a member of the family of sialic acids, each of which is

acylated at a different site. These compounds are usually found as terminal carbohydrate

residues of oligosaccharide side chains of glycoproteins, glycolipids, or, less frequently, of

glycosaminoglycans. The carbons and nitrogens in NANA come from N-acetyl mannosamine

and phosphoenolpyruvate (an intermediate in the glycolytic pathway). Before NANA can be

added to a growing oligosaccharide, it must be converted into its active form by reacting with

cytidine triphosphate (CTP). The enzyme CMP-NANA synthetase catalyzes the reaction. This is

the only nucleotide sugar in human metabolism in which the carrier nucleotide is a

monophosphate.

B. Synthesis of acidic sugars

D-Glucuronic acid, whose structure is that of glucose with an oxidized carbon 6 (CH2OH

COOH), and its C-5 epimer, L-iduronic acid, are essential components of glycosaminoglycans.

Glucuronic acid is also required in detoxification reactions of a number of insoluble compounds,

such as bilirubin, steroids, and several drugs, including morphine. In plants and mammals (other

than guinea pigs and primates, including humans), 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.

2. Glucuronic acid:

Glucuronic acid can be obtained in small amounts from the diet. It can also be obtained from

the intracellular lysosomal degradation of glycosaminoglycans, or via the uronic acid pathway.

The end product of glucuronic acid metabolism in humans is D-xylulose 5-phosphate, which can

enter the

hexosemonophosphate pathway and produce the glycolytic intermediates glyceraldehyde 3-

phosphate and fructose 6-phosphate.


The active form of glucuronic acid that donates the sugar in

glycosaminoglycan synthesis and 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 carbohydrate chain. Uronosyl 5-epimerase causes epimerization of the D-to the L-sugar.

C. Synthesis of the core protein

The core protein is synthesized on and enters the rough endoplasmic reticulum (RER). The

protein is then glycosylated by bound glycosyl transferases located in the Golgi.

D. Synthesis of the carbohydrate chain

Carbohydrate chain formation begins by synthesis of a short linkage region on the core protein

on which carbohydrate chain synthesis will be initiated. The most common linkage region is

formed by the transfer of a xylose from UDP-xylose to the hydroxyl group of a serine (or

threonine) catalyzed by xylosyl transferase. Two galactose molecules are then added,

completing the trihexoside. This is followed by sequential addition of alternating acidic and

amino sugars, and epimerization of some D-glucuronyl to L-iduronyl residues.


E. Addition of sulfate groups

Sulfation of the carbohydrate chain occurs after the

monosaccharide to be sulfated has been incorporated into the

growing carbohydrate chain. The source of the sulfate is 3'-

phosphoadenosyl-5'-phospho-sulfate (PAPS, a molecule of

AMP with a sulfate group attached to the 5'-phosphate).

Sulfotransferases cause the sulfation of the carbohydrate

chain at specific sites. PAPS is also the sulfur donor in

glycosphingo lipid synthesis.

A defect in the sulfation of the growing glycosaminoglycan

chains results in one of several autosomal recessive disorders

(chondrodystrophies) that affect the proper development and

maintenance of the skeletal system.

IV. DEGRADATION OF GLYCOSAMINOGLYCANS

Glycosaminoglycans are degraded in lysosomes, which contain

hydrolytic enzymes that are most active at a pH of

approximately 5. Therefore, as a group, these enzymes are

called acid hydrolases. The low pH optimum is a protective

mechanism that prevents the enzymes from destroying the

cell should leakage occur into the cytosol where the pH is

neutral. With the exception of keratan sulfate, which has a

half-life of greater than 120 days, the glycosaminoglycans have

a relatively short half-life, ranging from about 3 days for

hyaluronic acid to 10 days for chondroitin and dermatan

sulfate.

A. Phagocytosis of extracellular glycosaminoglycans

Because glycosaminoglycans are extracellular or cell-surface

compounds, they must first be engulfed by an invagination of the cell membrane

(phagocytosis), forming a vesicle inside of which the glycosaminoglycans are to be degraded.

This vesicle then fuses with a lysosome, forming a single digestive vesicle in which the

glycosaminoglycans are efficiently degraded.


B. Lysosomal degradation of glycosaminoglycans

The lysosomal degradation of glycosaminoglycans requires a large number of acid hydrolases

for complete digestion. First, the polysaccharide chains are cleaved by endoglycosidases,

producing oligosaccharides. Further degradation of the oligosaccharides occurs sequentially

from the non-reducing end of each chain, the last group (sulfate or sugar) added during

synthesis being the first group removed.

MUCOPOLYSACCHARIDOSES

The mucopolysaccharidoses are hereditary diseases caused by a deficiency of any one of the

lysosomal hydrolases normally involved in the degradation of heparan sulfate and/or dermatan

sulfate. They are progressive disorders characterized by accumulation of glycosaminoglycans in

various tissues, causing a range of symptoms, such as skeletal and extracellular matrix

deformities, and mental retardation. Children who are homozygous for any one of these

diseases are apparently normal at birth, then gradually deteriorate. In severe cases, death

occurs in childhood. All are autosomal recessive diseases except Hunter syndrome, which is X-

linked. Incomplete lysosomal degradation of glycosaminoglycans results in the presence of

oligosaccharides in the urine. These fragments can be used to diagnose the specific

mucopolysaccharidosis by identifying the structure present on the nonreducing end of the

oligosaccharide, as that residue would have been the substrate for the missing enzyme.

Diagnosis is confirmed by measuring the patients cellular level of the lysosomal hydrolases .

Bone marrow and cord blood transplants have been used to treat Hurler and Hunter

syndromes. Here the transplanted macrophages produce the enzymes needed to degrade

glycosaminoglycans in the extracellular space. Enzyme replacement therapy (ERT) is currently

available for both syndromes. In addition to the degradation of glycosaminoglycans, lysosomal

endo - and exoglycosidases are also involved in the degradation of glycoproteins and

glycolipids. Deficiencies in these enzymes result in the accumulation of partially degraded

carbohydrates in the lysosomes, leading to cell and tissue damage.


From Marks:


From Garys notes:

The basal lamina is actually the basement

membrane i.e. layers of epithelial cells

resting on the extracellular matrix.

The production and assembly of all these

components is responsible for the correct

functioning of cells and tissues such as in

differentiation and motility, and the


maintenance of the tissue phenotype. Problems in some components have been implicated and

proven in some forms of disease as diverse as:

1. Muscular dystrophy

2. Dwarfism

3. Renal diseases associated with incorrect filtration

4. Lysosomal storage diseases (severe and rare)

Example:

Type IV collagen is a non-fibrillar, network-forming

collagen which forms part of the extracellular matrix. The

lattice, shown in part D in the diagram, provides structural

support to the basal lamina. The basic protomer retains a

carboxyl terminal globular domain (in the case of fibrous

collagens have this removed). Other important collagens

are transmembrane proteins, helping to secure the cell to

the extracellular matrix surrounding it.

Laminins


Laminins are a component of the basement membrane too. They are abundant in the basal

lamina. They bind to multiple components of the cell and for the extracellular matrix. They can

form long polymers bound by the short arms. They usually bind to collagen IV. Since laminin is

composed of three chains; there are the different possibilities of 5 alpha chains, 3 beta chains

and 3 gamma chains. There are 45 different laminins altogether but only 12 have been

discovered yet. Mutations in laminin, usually laminin 5 and laminin 6, cause the disease,

junctional epidermolysis bullosa; which consists of extreme blistering of the skin. This disease

can be fatal; JEB gravis. Other mutations, in laminin 2, cause congenital muscular dystrophy.

Fibronectin domains

It is similar to fibrillin in terms of multiple binding domains throughout the sequence. It looks

like a string of beads. It is found in the extracellular matrix and in the plasma. There are 20

different types produced by alternative splicing. Which splice sites are used is determined by

tissue, wound healing, development and oncogenesis. Loss of fibronectin from tumour cell

surfaces may help metastasis; as cells can then penetrate the extracellular matrix. Fibronectin s

capable of binding to fibrin, collagen, heparin and cell surfaces as weel as intracellular

components.

Note: PXSRN and RGD in the diagram above refer to amino acid codes.

Glycosaminoglycans

They are types of heteropolysaccharides, which means that they are complex

carbohydrates formed by combining carbohydrates with non-carbohydrates or carbohydrate

derivatives; examples include pectin, lignin, glycoproteins, glycolipids, and

mucopolysaccharides.


They are made up of acid-amino sugar residues in the repeat unit. They

are negatively charged at physiological pH and they bind a lot to water.

These glycosaminoglycans exhibit resilience due to its hydrophillicity. This

is the ability to return to its original form after being stretched or

compressed. The biophysical properties are more important then

biochemical properties. They exibit both viscous and elastic properties

therefore referred to as viscoelastic.


There are several classes of glycosaminoglycans:

1. Hyaluronate

They are not sulfated. They form large polymers.

They act as lubricant and shock absorber. Found in

synovial fluid, vitreous humour, umbillical cord and

cartilage.

2. Chondroitin sulfates

They are the most abundant

glycosaminoglycans. They are

found in aggregates such as

proteoglycans and with

hyaluronate. Found in cartilage,

tendons, ligaments, aorta. They

can bind to collagen.

3. Dermatan sulfates

Found in skin, blood vessels, and heart valves. L-Iduronate

is an important component; mostly its carboxylic acid

group at carbon 6.


4. Keratan sulfate

They are very heterogeneous as they contain other

monosaccharides. Found in connective tissues,

proteoglycans and the cornea. Keratan is also

found in bones. It is made also in the brain in

response to damage. Galactose is an important

component.

5. Heparin

They are highly sulfated. They are found

intracellularly such as in mast cells. They interact

with antithrombin III. Heparin activates

antithrombin III which inactivates thrombin

therefore an anticoagulant action. Antithrombin III

is a serpin. It does not dissolve clots. It binds to

various proteins electrostatically when it is found

on the outer surface of some cells.

Most glycosaminoglycans are sulfated, except hyaluronate. Errors in the sulphation step in the

synthesis of these glycosaminoglycans can result in chondrodystrophies which are autosomal

recessive disoders of cartilage development. This will lead to an error in the development of the

skeletal system. Synthesis occurs in the golgi so that they can be exported from the cell.

Their negative charges help to maintain an extended conformation of the polysaccharides and

help polysaccharide molecules to slide pass each other. This gives mucus and synovial fluid their

viscous and lubricating properties.

Proteoglycans

Proteoglycans are any of a group of

polysaccharide-protein conjugates

present in connective tissue and

cartilage, consisting of a polypeptide

backbone to which many

glycosaminoglycan chains are

covalently linked; they form the


ground substance in the extracellular matrix of connective tissue and also have lubricant and

support functions. Negative charges help to maintain the structure by their mutual repulsion.

Linkage Region of Proteoglycans

O-linked sugars join the

glycosaminoglycans to the

core protein covalently.

Various protein cores imply

that they are not just a

scaffold for the

glycosaminoglycans but play

an essential role in cell

growth and differentiation.

The core proteins have

domains with particular

biological activities.

In this diagram above what is shown is non-covalent bonds joining the core protein to

hyaluronic molecule to form an aggregate. Link protein help to maintain the structure. This is

about 10 million daltons and contains about 10000 neg. charges. The charges require counter

ions which help draw water into the extracellular matrix. The osmotic pressure which results

then increases the stiffness. Hyaluronic acid is synthesised by the cells, the plasma membrane

not the ER; which then extrudes into the extracellular matrix space directly as its being made.


Proteoglycan aggregates: Aggrecan

The bottlebrush

structure form the

aggrecan.

Extracellular Matrix

Proteins of the extracellular

matrix bind other components

as well as the cells embedded

within it. They act very like

reinforced concrete in

buildings with collagen acting

as the steel reinforcing bars

while aggrecans are the

cement. Integrins are the

major cell surface proteins

which bind to the ECM

components.


Cartilage

Tissue remodeling:

The reorganization

or renovation of

existing tissues.

This process can

either change the

characteristics of a

tissue such as in

blood vessel

remodeling, or

result in the

dynamic

equilibrium of a

tissue such as in

bone remodeling.

This is an example of how extracellular components are continuously synthesized and

degraded. In addition, matrix metalloproteinases, which are zinc-containing proteases, are used

to degrade proteins of the ECM. One class of matrix metalloproteinases are collagenases. These

are required for tissue remodeling. Many of the proteins of the ECM have domains

corresponding to growth factors, which can be released by the action of metalloproteases. This

then encourages tissue growth. Metalloproteases are therefore vital for the on-going process of

tissue remodeling.

Lysosomal degradation of

glycosaminoglycans.

This diagram shows an example of a group

of degradative enzymes of

glycosaminoglycans. This is showing

degradation of dermatan sulfate.


Mucopolysaccharides

They are any of a group of complex polysaccharides composed of repeating units of two sugars,

one of which contains an amino group.

Degradation of Heparan sulfate


Note: This is the same diagram from Lippincotts found further up in the notes. Just to show

that GJH put it in his notes and therefore emphasize it.

Objectives

1. Know and name the components of the extracellular matrix.

Pages: 1,4,9,12,15,16,17,19,24

2. Describe the general structure of glycosaminoglycans and proteoglycans

Pages: 2,3,4,12,20,21,22,23,24

3. Explain the biophysical properties of glycosaminoglycans and proteoglycans.

Pages: 3-9, 13,14,20, 21-26

4. Know how enzyme defects result in mucopolysaccharidoses

Pages: 26,27

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