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- 36

- Rawan Almujaibel

محمود الحربي-

- Nafith

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The Extracellular Matrix

Extracellular Matrix is a fluid that contains proteins secreted by cells and

contains high carbohydrate content and it fills the spaces between cells

and binds tissues together.

There are types of proteins in the ECM called linker proteins not

secreted by the cells. Their job is to link the proteins in the solution to

the cells to form a network of proteins. This network connects cells

together, it connects proteins together and it connects cells to proteins.

This network is what makes the extracellular matrix a jell-like structure.

The extracellular matrix has two main components: the basal lamina and

connective tissue

Basal laminae: it is found beneath the endothelium and the epithelium.

These two types of cells rest on the basal lamina FROM SLIDE: It is a

Thin, sheet-like, structure upon which layers of epithelial cells rest. Also,

it is surrounds muscle cells, adipose cells, and peripheral nerves.

Connective tissue: It is loose network of proteins and carbohydrates

underneath epithelial cell layers where fibroblasts are distributed.

The connective tissue has proteins which help in connecting the cells

that were already in it to the basal laminea or they connect cells to each

other or even with other proteins.

If you have a close look on the connective tissue in bones, cartilage or

tendons, will find they are closely related to each other. However, the

type of secretions and type of deposited materials inside each of them

are different, so that makes them have their own unique properties.

For example: if you compare bones and tendons, you will find that

bones are harder than tendons due to deposition of calcium in bones.

Also, tendons are made mainly out of fibrous proteins which make them

softer than bones. Cartilage is made of high concentration of

carbohydrates (which makes it more viscous and allows it to return to its

normal shape if it is changed).

So the property of the tissue in general depends on the secretion and

the deposition of substances in the extracellular matrix.

FROM SLIDE:

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Matrix Structural proteins

-Tough fibrous proteins embedded in a gel-like polysaccharide ground

substance.

- Basal laminae is formed of matrix components that differ from those

found in connective tissues.

• Tendons: mainly fibrous proteins

• Cartilage: high concentration of polysaccharides (gel)

• Bone: hardened by deposition of calcium phosphate crystals.

There are 3 components of the ECM:

1) The matrix proteins

2) Polysaccharides

3) Adhesion proteins (link the matrix proteins to each other and link the

cells to the matrix as well); different tissues have different types of these

proteins

Now we are going to talk about the 2 main proteins that are secreted in

the extracellular matrix . . .

1- Collagens (is a glycosylated protein- its function to increase the

viscosity in the extracellular matrix) :

• The most abundant proteins in mammals (25% of the total

protein mass).

• Long, stiff, triple-stranded helical structure made of three α

chains.

(Their shape is triple-stranded; has 3 polypeptide chains wrapped

around each other. The amino acid structure is triplicate and the

glycine is repeated every 3rd amino acid, so the content of the

glycine is 1/3 of all amino acid in collagen).

• A basic unit of mature collagen is called tropo-collagen.

(Collagen is produced as a pro-collagen by fibroblast cells that are

in the extracellular fluid, so at the end it becomes mature to be

tropo-collagen. When we have the mature form of collagen, it will

attach and link with other collagen to form collagen fibrils then it

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forms fibres to be more condense and tough which can be seen

under light microscope).

Pro-collagen (immature) Tropo-collagen (mature) Fibrils

(more complex) Fibres (more condense and tough).

• Contents are rich in glycine (33%), proline (13%), and hydroxyl-

proline (9%).

(proline can be hydroxilated by enzyme called prolil-hydroxylase

and their co-factor is Vitamin C. If we don't have Vitamin C the

helical content of the collagen will be damaged. Also proline can

be modified by endoplasmic reticulum and in Golgi).

• Contains hydroxylysine (attachment of polysaccharides).

(Lysine can be hydroxylated in the collagen to become

hydroxylysine. The hydroxylysine is attachment for

polysaccharides).

• Cross-linking of chains via lysine and aldolysine via the action of

lysyl oxidase.

(The hydroxylysine can be oxidised by an enzyme called lysyl

oxidase which transfers it into aldehyde form "aldolysine", that

permits cross-linking in between collagen fibres and therefore we

will get very strong collagen).

Types of collagens:

Until now it is reported 28 types of collagens produced by very

different genes. Collagens are classified according to their types

(type I, type II… type 28)or according to their adopted shape.

• 30 collagen genes that form more than 25 different types of

collagen that resist tissue stretching.

• Types:

- Fibrillar collagens (I, II, III, V, XI) – they look like fibres.

- Fibril-associated collagens (IX, XII, XIV, XIX, XXI) – they are

fibers in their shape but they have very small piece of collagen

that help to associated with other collagen fibres, or with

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network of the collagen, or with basal laminea, or with other

proteins.

- Network-forming collagens – they are forming network form,

not fibre form.

- Anchoring fibrils.

- Transmembrane collagens.

All these types are named according to the adopted shape of collagen.

1-Types of fibrillar collagens:

NOTE: one of the most important properties of fibrillar is very

rigid in their structure- we can find it in bones.

• Type I (90% of the total collagen component in the body): most

connective tissues (Long, aligned in parallel to each other, and

rigid (fit to be in bone structure)).

• Type II: cartilage and vitreous humor. It also makes fibres but

they are aligned randomly unlike type 1, which permits it from

adopting the rod-like structure (important in cartilage). However

that does not mean it is not rigid.

FROM SLIDE:

Smaller in diameter than type I and oriented randomly in the

viscous proteoglycan matrix

Rigid macromolecules, but compressible (to resist large

deformations in shape and absorb shocks)

• Type III: extensible tissues (skin and lung)

• Type XI: cartilage

• Type XXIV: bone and cornea

• Type XXVII: eye, ear, and lung.

NOTE: Each type has tissue localization; therefore these specific

areas apply and help in their functions.

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Another note: the doctor said that he put these as examples for

you to know that each type of collagen has its own tissue

localization, so I don’t know if they are for memorization or not.

FROM SLIDE:

Assembly of fibrillar collagens

• After secretion, they assemble into collagen fibrils (thin

structures visible in electron micrographs).

• Collagen fibrils aggregate into collagen fibres (larger, cable-like

bundles seen in the light microscope).

• Fibril-associated collagens: Collagen IX and XII: link fibrils to one

another and to other ECM components

• Network-forming collagens: Types IV: basal laminae constituent

• Anchoring fibrils: Associate network-forming collagens to fibrillar

collagens

How collagen is synthesised?

- Firstly it is produced and modified in the endoplasmic

reticulum then modification is completed in the Golgi.

NOTE: N-terminus and C-terminus stay on collagen even after

the modification has happened. They will be secreted outside

of the cells while they are on immature collagen (C- and N-

terminuses makes the collagen called pro-collagen and after

they are cut outside the cell, they are called tropo-collagen).

N- & C- terminus will be on the collagen until forming the 3

polypeptide chains which are not wrapped around each other

well. When they reach this stage they will get out of the cell in

this form (3 polypeptide chains not wrapped around each

other well). Afterward, there are N- & C- peptidase enzyme

outside the cells called procollagen peptidases, their function is

to cut the terminuses. The presence of terminuses in the

collagen (at the ends on the rod like structures) prevents the

collagen from aggregating and attaching to each other inside

the cells. That is the reason we have collagen free within the

cells.

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- After cutting the terminuses, they will be able to crosslink each

other with aid of lysyloxidase enzyme (lysyloxidase enzyme is

secreted out from the cell "their localization is out of the cell, it

won't work inside the cell).

FROM SLIDE:

Synthesis of collagen

• Individual collagen polypeptide chains are synthesized into

the endoplasmic reticulum (ER) as pro-alpha chains

• In the ER, selected prolines and lysines are hydroxylated and

some of the hydroxylysines are glycosylated

• One alpha chain combines with two others forming

procollagen

• During or following exocytosis, extracellular enzymes, the

procollagen peptidases, remove the N-terminal and C-terminal

propeptides forming tropocollagen (or simply collagen)

• Excision of both propeptides allows the collagen molecules to

polymerize into normal fibrils in the extracellular space.

What makes other type of collagens adapt different forms and shapes?

We all know that the structure of the collagen is composed of 3

polypeptide chains wrapped around each other in a helical manner. This

structure is not applied at the whole length of the collagen molecule.

For example, some types are helical in their shape which adapts a rod-

like structure which gives the proteins its strength, and then they are

spanned by amino acids residues which separate the helical region and

followed by another helical region is separated by another amino acid

and so on. This makes the protein not to be rigid in the whole structure,

you will find some areas are less rigid and some flexible as well. Because

of this, there are different types and shapes of collagen.

FROM SLIDE:

Fibrillar vs. network-forming collagens

• The structure of network-forming collagens is interrupted by one or

two short non-helical domains, which makes the molecules more flexible

than fibrillar collagen molecules.

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• Network-forming collagens are not cleaved after secretion and

therefore retain their pro-peptides.

• Network-forming collagens do not aggregate with one another to form

fibrils in the extracellular space.

NOTE the Dr. skipped the information (below), I am not really sure if

we are going to be asked about them:

Premature assembly is inhibited

• The potentially catastrophic assembly of fibrils within the cell does not

occur because:

• The pro-peptides inhibit fibril formation

• Lysyl oxidase, which catalyzes formation of reactive aldehydes, is an

extracellular enzyme.

Collagen-related diseases:

Collagen is highly cross-linked in tissues where tensile strength is

required such as Achilles tendon. If cross-linking is inhibited, the tensile

strength of fibres is greatly reduced, collagenous tissues become fragile,

and structures tend to tear (skin, tendon, and blood vessels).

1-Scurvy:

It is an acquired disease (person can acquired it through the life)

It happens due to deficiency of Vitamin C it affects lysyloxidase &

prolilhydroxylase enzymes ( Vit C is important in hydroxylation process).

As a result of this:

-The helical formation of the collagen will be affected; therefore with

the temperature inside the body the helical formation is changed.

-lysyloxidase won't work properly; therefore the cross-linking won't be

proper, so the collagen will be very weak and arteries wall will be weak

as well, which makes the person to bleed very easily. Also, type 1 will be

affected as well leading to fractures.

From SLIDE:

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Deficiency of vitamin C results in insufficient formation of hydroxyl-

proline and, hence, poor synthesis of collagen, formation of unstable

triple helices.

Non-hydroxylated pro-collagen chains are then degraded within the cell.

Symptoms: skin and gum lesions & weak blood vessels.

2-Osteogenesis imperfect (OI) (Brittle-bone disease):

It is a defect in the formation of collagen caused by genetic

mutations.

It is rare disease caused by imperfect bone formation. A genetic disorder

of several forms that cause fragile, soft, brittle, and easily broken bones

There are four types of Osteogenesis Imperfecta designated as type I

through type IV

• Type I: the mildest form of the condition.

• Type II: the most severe form that results in death in uterus or shortly

after birth.

• Milder forms generate a severe crippling disease.

3 types out of 4 (type 1, 2, and 4) are autosomal dominant which you

need only one copy of the altered gene is sufficient to cause the

condition.

People who get this disease are more likely to be exposed to fractures,

due to weak collagen. These patients rely on the minerals deposited in

the bone to endure forces, and minerals alone are not enough to protect

the bone. So these minerals need support, which is given to them by

collagen. That is why defective collagen causes increases bone fractures.

Osteogenesis Imperfecta patients get blue sclera because the collagen

inside the eyes is very weak and is separated from each other, so the

venous blood behind the sclera appears and gives the patient the blue

appearance in the eyes.

FROM SLIDE:

Mutations of OI

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• Mutations in COL1A1 (it is mainly happened in collagen TYPE 1) and

COL1A2 genes that interfere with the assembly of type I collagen.

• Defective collagen weakens connective tissues, particularly bone,

resulting in the characteristic features of OI.

3-Chondrodysplasias:

It is a disease caused by mutations that affect chondrocytes function of

making type II collagen, characterized by abnormal cartilage (because

cartilage has a lot of type2 collagen), which leads to bone & joint

deformities.

The whole cartilage in the body will be affected, and any problem that is

associated with cartilage problems are possible to arise. Cartilage won't

be produced properly, type 1 or 2 collagen won't be functioning properly

and the associated carbohydrates in the cartilage won't move well.

Some patients can't walk well because their cartilage will be shortened

little bit, so they are not compressed well. Then over period of time we

will find degeneration (problems in movement of the whole body due to

the affect in the joints).

4-Ehlers-Danlos Syndrome:

It is a disease affecting the collagen formation. It is a heterogeneous

group of disorders that affect the skin, bones, blood vessels, and other

organs. Mutations happen in many types such as type I, III, or V collagen

and affect the synthesis of collagen. Also, it happens in processing

enzymes like pro-collagen N-peptidase, or lysyl-hydroxylase.

NOTE: it is a multifactorial disease. Strong collagen gives rigidity and

weaker collagen gives more flexibility.

Due to weakness of the collagen it will affect the joints (which are

covered by tendons) and make them very flexible. Also, the skin will be

hyper-extensible.

FROM SLIDE:

The signs and symptoms vary from mild to life-threatening.

All result from defects in collagen synthesis and/or processing.

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Major manifestations are skin fragility & hyper-extensibility & joint

hypermobility.

• The most clinically important mutations are found in the gene of type

III collagen.

• Since type III collagen is a major component of arteries, mutations

affecting type III collagen result in fragile blood vessels.

Other symptoms include stretchy skin & hypermobile joints.

..................

Now we are going to talk about the second most important protein that

is secreted in the extracellular matrix...

2-Elastin (it is not a glycosylated protein):

- The main component of elastic fibres is elastin. It is highly

hydrophobic and rich in proline and glycine. It contains some

hydroxyproline, but no hydroxylysine.

Formation of the elastic network:

1- Secretion of tropo-elastin (same as tropo-collagen).

2- Assembly into elastic fibers.

3- The cross-linking via lysines (through lysyloxidase).

Elastin structure:

• The elastin protein is composed largely of two types of short segments

that alternate along the polypeptide chain:

• Hydrophobic segments, which are responsible for the

elastic properties of the molecule; and

• Alanine- and lysine-rich a-helical segments, which form

cross-links between adjacent molecules.

Function of elastic fibre:

• Elastin is the dominant Extracellular matrix protein in arteries.

• The normal elasticity of an artery restrains the proliferation of these

cells.

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• Abnormal or deficiency of the elastin results in excessive proliferation

of smooth muscle cells in arterial walls and narrowing of the aorta.

Elastin Tissue:

Elastin tissue is covered with a sheath of microfibrils (they are fibers that

have distinct glycoproteins, including the large glycoprotein fibrillin,

which binds to elastin and is essential for the integrity of elastic fibers).

When we have a problem in fibrillin such as mutation it will cause a

disease called Marfan's Syndrome.

Elastin-related disease:

1-Marfan's Syndrome:

This disease is happened due to mutated in fibrillin (so when the sheath

is not covering the elastin, the tissue will be very weak. For example, the

elasticity of the arteries will be decreased due to loss of the microfibrils

sheath so these arteries will not be able withstand the pressure that

comes from the heart, this leads to rupture in the main arteries)

Clinical features (FROM SLIDE):

A tall, thin build; Long arms, legs, fingers, and toes and flexible joints;

Scoliosis, or curvature of the spine; A chest that sinks in or sticks out;

Crowded teeth; Flat feet

(This piece of information was in the slide, but not mentioned by the

Dr.).

2-Emphysema (destructive lung disease):

Is a dysfunctional α1- antitrypsin deficiency that leads to increased

activity of elastase and it is characterized by destruction of alveolar

walls. α1-antitrypsin main function is to inhibit the elastase enzyme

which breaks down elastin, so if there is a deficiency in this enzyme, the

elastase will not be inhibited and it will destroy elastin in the alveolar

walls. A mutation happens by changing Lysine to Glutamic acid which

causes misfolding, forming aggregates & block of ER export.

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Cigarette smoking also inactivates α1-antitrypsin by oxidizing essential

Met (359) residue, decreasing the enzyme activity by a factor of 2000

folds.

..................................................

Now will move to talk about Extracellular Matrix Polysaccharides...

Glycosaminoglycans (GAGs) (it is the main component in the

extracellular matrix):

It is polysaccharides of repeated disaccharides. One is either N-

acetylglucosamine or N-acetylgalactosamine (amino sugar- which is

modifies by amino group). The other is usually acidic and it is either

glucuronic acid or iduronic acid. The latter two are highly negatively

charged and are sulphated most of the time (not always sulphated). One

of the types of glycosaminoglycans is proteoglycans which have

carbohydrates as their main content.

Most of the Glycosaminoglycans exist as proteoglycans (Cell surface

proteins with either transmembrane domains (syndecans) or GPI

anchors (glycipans) interacting with integrins).

1. Hyaluronan acid (not a proteoglycan): anionic, non-sulphated

GAG distributed widely throughout connective, epithelial, and

neural tissues.

Aggregan as a molecule (an example of them):

It is a large proteoglycan (core protein) consisting of more than

100 chondroitin sulfate chains joined to a core protein. Multiple

aggregan molecules bind to long chains of hyaluronan that

become trapped in the collagen network, forming large

complexes in the Extracellular matrix of cartilage mainly.

Why Glycosaminoglycans is the main thing in the extracellular matrix?

Because their negativity is very high due to the huge number of the

repeating disaccharides (one of them amino sugar and the other is

sulphated acidic in nature most of the time), plus the negativity that we

find in the carbohydrates adding the sugars. Also, the shapes that they

adopt specifically the branches that it has, gives you high connectivity in

extracellular matrix higher than any carbohydrates.

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Now will move talking ABOUT Extracellular Adhesion proteins:

They link matrix proteins with one another and to the surfaces of cells.

The main protein is Fibronectin (secreted by fibroblasts):

It is the principal adhesion protein of connective tissues.

It is a dimeric glycoprotein that is cross-linked into fibrils by disulfide

bonds. It is secreted by the fibroblasts and binds to collagen and GAGs

that are already found in the extracellular matrix from one side. From

the other side they bind to transmembrane proteins (integrins) in the

plasma membranes of cells.

That’s how the network in the extracellular matrix is made. Firstly,

Collagen is secreted to give some strength to the extracellular matrix

which is not linked to anything. The GAGs are present and most of them

are attached to proteins (in a proteoglycan structures). Then helical

proteins attach to collagen in one side, GAGs in other side, and attach to

the cell as well through the integrin protein within the plasma

membrane (so the network is completed then).

Other main protein is laminin:

It found in basal laminae. It is T-shaped hetero-trimer with binding sites

for cell surface receptors, type IV collagen and perlecan. It has 3 chains:

A chain is in the centre and other 2 chains are B1 and B2 wrapped

around each other over the A chain.

Laminins are tightly associated with another adhesion protein, called

nidogen, which also binds to type IV collagen, all of which form cross-

linked networks in the basal lamina.

Cell-matrix interactions: Role of integrins

Any protein in the extracellular matrix will mediate its effect over the

cell through integrins. They are a dimer of 2 chains: α & β. The integrins

mediates the signals that are coming from outside the cell or going

outside from the cell (from nucleus to the cell membrane, extracellular

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matrix and so one). It binds to the cytoskeleton from inside, and binds to

the extracellular matrix from outside. Integrins work as dimers.

They are inactive at first then it will become active upon binding to

other molecules.

Integrins are a family of transmembrane heterodimers (α & β). They

anchor the cytoskeleton at focal adhesions and hemi-desmosomes.

What is focal adhesion?

When we talked about the cell and how it moves, we came across the

actin filaments they are polymerised in specific area in the cell. Why

does actin polymerize in that specific area? Because actin-binding

proteins are concentrated in that area. Why are actin-binding proteins

concentrated in that area? Because of signals that come from outside

the cell. These signals affect integrins by activating them, so integrins

localize at that area and actin-binding proteins bind to them. So this

complex recruits actin and ‘tells’ actin that it needs to polymerize in this

area. So actin polymerizes while it is bound to the actin-binding protein.

So focal adhesion is an area with collection of proteins.

FROM SLIDE: Development of focal adhesion:

1. Activation of integrin and binding to Extracellular matrix.

2. Recruitment of additional integrins forming focal complex.

3. Development of focal adhesion.

........................................................................

Cell to cell interaction

Cells interact with each other by junctions, for example by adherence

junction (like desmosomes), how do they interact by these junctions? ...

So, there are Actin filaments surround the cell from inside. They

communicate with the outer signals by attaching to cadheren ( a

transmembrane protein); and the cadheren is attached with another

cadheren from other cell and so on. So the interaction happens in this

way.

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Cell interaction devided into 2 types:

1- Homophilic interaction :(when the proteins within between the

cells are the same type we call this type as homophilic)

2- Heterophilic interaction : (when the proteins in between the cells

are different types we call this type of interaction is heterophilic)

We will talk about 2 of them ....

Selectins:

They are different types (all types called a group of selectins). It has a

role in leukocyte-endothelial cell interaction "attachment and squeeze

of leukocyte":

1-Rolling of leukocytes is mediated by selectin (L-selectin, P-selectin, and

E-selectin are attached to leukocyte, and then they facilitate the

attachment to the endothelium) .

2-Prior to invading a blood vessel, integrins are activated (the integren is

activated when the selectens and leukocyte are connected with each

other by sending signal to integren)

3-firm attachment allows invasion (when integren is activated it will

send signals to actine filaments to grow, then the leukocyte will squeeze

and attachment will occur).

Cadherins:

• Selective adhesion between embryonic cells and formation of stable

junctions between cells in tissues.

Types:

- Classical tissue cadherins

- E-cadherin: epithelial cells

- N-cadherin: neural cells

- P-cadherin: placental cells

- Desmosomal cadherins

- 7-Transmembrane cadherins

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As you can see there are lots information in this sheet I advise you to go

back to the slide and refer to the pictures and figures there... Sorry for

any mistake.

Best of luck...