ECM

Question 1

What is the extracellular matrix?

What types of components does the ECM comprise of?

Answer 1

Network of proteins + carbohydrates filling spaces between cells

ECM comprises both fibrillar and non-fibrillar components

Question 2

What are the key functions of the ECM?

Answer 2

Extracellular matrix functions:
-provides physical support

-determines mechanical and physicochemical properties of the tissue

-affects growth, adhesion & differentiation status of the cells and
tissues it interacts with

-essential for development, tissue function & organogenesis

Question 3

What is connective tissue?

What are the components of connective tissues?

What do matrix components interact with?

Answer 3

Connective tissue = ECM + component cells

Components of connective tissues:

-collagens: type I, II, III (fibrillar) 
                    type IV (basement membrane) 

-multi-adhesive glycoproteins: fibronectin, fibrinogen
Laminins (basement membrane)

-proteoglycans: aggrecan, versican, decorin
Perlecan (basement membrane)

Matrix components interact with specific cell surface receptors

Question 4

Describe the varied properties of the following types of connective tissues:

• Tendon & skin
• bone
• cartilage

Answer 4

Tendon & skin: tough & flexible

Bone: hard & dense

Cartilage: resilient & shock absorbing

Question 5

What types of human disorders result from ECM gene mutations? List some disorders caused by the 2 types of gene mutations that involve the ECM

Answer 5

1. Gene mutations affecting matrix proteins
   Eg:
   -osteogenesis imperfecta (type I collagen)
   -Marfans syndrome (Fibrillin 1)
2. Gene mutations affecting ECM catabolism [aka mucopolysaccharidoses (MPSs), inability to degrade GAGs]
   Eg:
   -hurlers syndrome (L-alpha-iduronidase)

Question 6

What other types of disorders can be caused by ECM abnormalities?

Answer 6

Fibrotic disorders due to excessive ECM deposition
Eg:
Liver fibrosis - cirrhosis
Kidney fibrosis - diabetic nephropathy
Lung fibrosis - idiopathic pulmonary fibrosis (IPF)

4. Disorders due to excessive loss of ECM
   Eg osteoarthritis

Question 7

What are collagens?

Answer 7

Family of fibrous proteins, major proteins in bone, tendon and skin
Most abundant proteins in mammals (25% of protein mass)

Question 8

Describe the alignment of collagen fibrils in the skin, mature bone and cornea

What do these tissues resist?

Answer 8

Skin: successive layers of collagen nearly at right angles to each other

Mature bone and cornea: same arrangement (successive layers of collagen at right angles to each other)

These tissues resist tensile force in all directions

Question 9

Molecular arrangements of collagen fibres:

How many collagen types exist in humans?
How many genes encoding collagens?m
How many alpha chains does each collagen molecule comprise of? What does this form?

Number of chains in type 1, 2 and 3 collagen?

Answer 9

28 types of collagens
42 genes encoding collagens
Each collagen molecule comprised of 3 alpha chains -> triple helix

Type 1 collagen: chains from two different genes
Type II and III collagen only have one chain type

Question 10

What type of helix structure does collagen have? What type of chains form this structure? And what amino acid occupies every 3rd position, and why?

What components make up the repeating structure?
How long are chains in fibrillar collagens?

Answer 10

Three alpha chains form a stiff triple helix
In fibrillar collagens, each alpha chain is ~1000 amino acids (forms left handed helix)
Alpha chains consist of a characteristic gly-x-y repeat (gly-glycine, x - usually proline, y - usually hydroxyproline)

Every third position in the alpha chains is occupied by glycine, bc glycine is small enough to occupy the interior (H side chain)

Question 11

Collagen biosynthesis:

What is the step that occurs to form collagen from pro collagen in the case of fibrillar collagen, that doesn’t happen in most other types of collagen?

Answer 11

All newly synthesised collagen chains have non collagen it’s domains at N- and C- termini.

To form collagen from pro collagen, the N and C termini are removed after secretion in the case of fibrillar collagens. But remain part of the collagen in most other types.

Question 12

What type of cross links are formed in collagen and between which residues?

What does cross linking provide?

What does the type and extent of cross link depend on?

Answer 12

Covalent cross links are formed in collagens, lysine and hydroxy-lysine residues are involved.

Cross linking provides tensile strength and stability

The type and extent of cross links is tissue specific and changes w age

Question 13

What do prolyl and lysyl hydroxylases require to form interchain hydrogen bonds?

What else are lysine and hydroxylysine modified in the formation of? When does this take place?

What causes scurvy?

Answer 13

Prolyl and lysyl hydroxylases require Fe2+ and vitamin C, which contributes to interchain hydrogen bond formation

Lysine and hydroxylysine are also modified in the formation of covalent cross linkages. Takes place after the collagen has been secreted.

Vitamin C deficiency results in underhydroxylated collagens —> tissue instability (scurvy)

Question 14

What are ehlers-Danlos syndromes (EDS)? What are the symptoms?

The disorders arising due to mutations in collagen negatively affect what?

Answer 14

Ehlers-Danlos syndromes (EDS) = group of inherited connective tissue disorders, symptoms include stretchy skin and loose joints.

Disorders arising due to mutations in collagen negatively affect:

• collagen production
• collagen structure
• collagen processing

Question 15

What types of collagen regulate the organisation or collagen fibrils?

What type of collagen is type IV collagen? Where is it present?

Answer 15

Fibril associated collagen (eg type IX and XII) associate with fibrillar collagens and regulate the organisation of collagen fibrils

Type IV collagen is a network-forming collagen, is present in all basemen membranes, but molecular constitution varies from tissue to tissue.

Question 16

What is the role of basement membranes in relation to tissue function?

What are basement membranes? What do they form in the kidney?

Answer 16

Basement membranes regulate tissue function. 
Basement membranes (/basal laminae) are flexible, thin mats of extracellular matrix underlying epithelial sheets & tubes. BMs surround muscle, peripheral nerves and fat cells. Contain distinct repertoire of collagens, glycoproteins & proteoglycans. 

Basement membranes form a key part of the filtration unit as the glomerular basement membrane (GBM) in the kidney.

Question 17

What is diabetic nephropathy?

What is Alport syndrome? List symptoms associated.

Answer 17

Diabetic nephropathy is a disorder where there’s an accumulation of extracellular matrix —> leading to thickened basement membrane. This restricts renal filtration + can lead to renal failure.

Alport syndrome - mutations in collagen IV result in abnormally split and laminated GBM, which is associated with a progressive loss of kidney function + hearing loss.

Question 18

What are elastic fibres important for?

Why are collagen and elastic fibres often interwoven?

What does the core of elastic fibres consist of?

Answer 18

Elastic fibres are important for the elasticity of tissues, like skin, blood vessels and lungs.

Collagen & elastic fibres are interwoven to limit the extent of stretching.

Elastic fibres consist of a core made up of the protein elastin, and microfibrils rich in the protein fibrillin.

Question 19

What are mutations in the protein fibrillin associated with? What can this predispose individuals to?

Answer 19

Mutations in the protein fibrillin are associated w marfans syndrome. Marfans syndrome has effects on the skeletal, ocular & cardiovascular systems. Ppl can be predisposed go aortic ruptures.

Marfans syndrome manifestations inc arachnodactyly - spider like fingers

Question 20

What two types of segments does elastin consist of and how are they arranged?

What types of side chains in elastin are covalently cross linked?

Answer 20

Elastin (protein) consists of alternating hydrophobic and alpha helical regions along the polypeptide chain.

The alpha helical regions are rich in alanine and lysine. Many lysine side chains are covalently cross linked

Question 21

What does it mean that ECM proteins have modular architecture?

How are ECM proteins multifunctional?

Describe some qualities of large modular proteins

Answer 21

ECM proteins have modular architecture and as a result most ECM proteins are very large, they’re composed of protein domains of 50-200 amino acids.

The multi functionality of ECM proteins is a result of their modular structure.

Many large modular proteins are multi-adhesive, binding various matrix components & cell surface receptors

Question 22

What are laminins? What types of chains do they consist of and what shaped molecules does this form?

Describe the size/weight of laminins

What type of cell surface receptors can laminins interact with?

Describe the interaction of laminins with the basement membrane matrix and it’s components

What inherited diseases are associated with mutations in specific chains of laminins? What are symptoms of congenital muscular dystrophy?

Answer 22

Laminins are multi-adhesive heterortrimeric proteins. Laminins have an alpha, beta and gamma chain which form a cross shaped molecule.

Laminins are v large proteins, each chain has a molecular weight of between 160-400 KDa.

As laminins are multi-adhesive proteins, they can interact w several cell surface receptors inc integrins & dystroglycan.

Laminins can self associate as part of the basement membrane matrix, but also interact w type IV collagen, nidogen & proteoglycans.

Inherited disorders caused by mutations in specific laminin chains: muscular dystrophy & epidermolysis bullosa.

Congenital muscular dystrophy can be caused by absence of alpha2 chain in laminin 2. Symptoms - hypotonia (abnormally decreased muscle tension), generalised weakness & deformities of the joints.

Question 23

What are fibronectins? Where are they found?

Describe the two different states fibronectins can exist in

Detail how the different types of fibronectins arise.

Describe the molecular structure of fibronectins

What components of the cell do fibronectins interact with? What do fibronectins regulate? And what is their role in haemostasis?

What types of receptors provide the link between the matrix and cytoskeleton?

Answer 23

Fibronectins = family of closely related glycoproteins of the extracellular matrix. Also found in body fluids and are multi-adhesive.

Fibronectins can exist either as insoluble fibrillar matrix or as a soluble plasma protein.

The different types of fibronectins are derived from a single gene, with alternate splicing of mRNAs giving rise to the diff properties.

Laminins are made up of a large multi domain molecule linked together by disulphide bonds.

Fibronectins interact with cell surface receptors & other matrix molecules. They play an important in regulating cell adhesion & migration in processes like embryogenesis & tissue repair.

Fibronectins are important for wound healing + promote blood clotting.

Integrin receptors at the cell surface provide linkage between matrix and cytoskeleton.

Question 24

What are proteoglycans? What type of chains are they covalently attached to?

What type of repeating units are these chains made up of? Why do many of these chains carry a high negative charge? What does this lead to in the ECM?

Answer 24

Proteoglycans are core proteins to which are covalently attached one or more glycosaminoglycan (GAG) chains.

GAG chains are made up of repeating disaccharide units, with one of the two sugars being an amino sugar.

Many GAGs are sulfated or carboxylated and therefore carry a high negative charge. The high negative charge attracts a cloud of cations inc Na+, leading to large amounts of water being sucked into the extracellular matrix.

Question 25

List 4 types of proteoglycan families and describe what they are grouped based on

Answer 25

Proteoglycan families are grouped based upon their structural & functional characteristics:

• basement membrane proteoglycans
• aggregating proteoglycans
• small leucine-rich proteoglycans
• cell surface proteoglycans

Question 26

Cartilage has a matrix rich in collagen w large quantities of GAGs trapped in the meshwork, so how is the balance of swelling pressure negated?

GAG chains are grouped into 4 main groups according to the repeating disaccharide unit, what are the 4 groups?

Answer 26

The swelling pressure in cartilage is negated by the tension in the collagen fibres, this generates great tensile strength.

4 main GAG chains (according to the repeating disaccharide unit):

• hyaluronan
• chondroitin sulfate and dermatan sulfate
• heparan sulfate
• keratan sulfate

Question 27

What is hyaluronan spun out directly from?

Where are all the other GAGs synthesised and attached to their core proteins?

Answer 27

Hyaluronan is spun out directly from an enzyme embedded in the plasma membrane.

All other GAGs are synthesised and attached to their core proteins in the endoplasmic reticulum & Golgi apparatus inside the cells.

Question 28

Where is hyaluronan/hyaluronic acid found?

Why is hyaluronan distinct from the other GAGs?

Why can hyaluronan create molecules or enormous sizes that can occupy a relatively large volume?

Describe the viscosity of hyaluronan and where it’s found in the eye and joints.

Answer 28

Hyaluronan is found in the extracellular matrix of soft connective tissues.

Hyaluronan is distinct from the other GAGs as it’s simply a carbohydrate chain without a core protein. It’s unsulfated & made up of repeating disaccharides.

Hyularonan can undergo a very high degree of polymerisation, in the range of 10’000 disaccharides, creating large molecules.

Hyaluronan is typically of high viscosity. Found in the vitreous humour of the eye and in synovial fluid of joints. In the synovial fluid of joints hyaluronan plays a role in protecting the cartilaginous surface from damage.

Question 29

What is aggrecan a major constituent of?

Why do the GAGs in aggrecan have an increased negative charge? What does this lead to?

Why is aggrecan suited to resist compressive forces?

Answer 29

Aggrecan is a major constituent of the cartilage extraceullar matrix.

In aggrecan the GAGs are highly sulfated and have large numbers of carboxyl groups that increase their negative charge. This attracts cations like Na+ that are osmotically active. This leads to large amounts of water being retained by the highly negatively charged environment.

Under compressive load, the water is given up, but regained once load is reduced, making aggrecan in the cartilage matrix suited to resist compressive forces.

Question 30

What effect does osteoarthritis have on the ECM?

What happens to cartilage over the end of bones and to aggrecan?

Answer 30

Osteoarthritis is an erosive disorder which results in excessive extracellular matrix degradation

The cushioning properties of cartilage over the end of bones are lost. With increasing age, aggrecan is cleaved by aggrecanses & metalloproteinases. This results in a loss of aggrecan fragments to the synovial fluid.

Question 31

What do fibrotic diseases arise as a result of?

Answer 31

Fibrotic diseases arise as a result of an excessive production of fibrous connective tissue