Extracellular matrix (ECM)

Question 1

What is the ECM?

Answer 1

• Complex network of macromolecules (proteins and carbohydrates) deposited by cells ,made up of fibrillar and non-fibrillar components
• After being deposited it becomes immobilised outside the cells and it fills in the spaces between the cells
• ECM essential for development, tissue function and organogenesis
• Architectural (mechanical stability) and instructional roles (influences cell behaviour)
• Proteoglycans/collagen/glycoproteins/enzymes etc

Question 2

What is the function of the ECM?

Answer 2

• Provide physical support
• Determine the mechanical and physicochemical proper-ties of the tissue
• Influence the growth, adhesion and differentiation status of the cells and tissue with which it interacts

Question 3

Describe connective tissue

Answer 3

-Rich in ECM
-All contain distinct spectrum of:
1. Collagens: e.g. type I,II,III (fibrillar). Type IV (basement membrane)
2. Multi-adhesive glycoproteins: e.g. Fibronectin, 3. Fibrinogen, Laminins (basement membrane)
Proteoglycans: e.g. Aggrecan, Version, Decorin, Perlecan (basement membrane)
-Each matrix component is able to interact with the cellular components via specific cell surface receptors

Question 4

What is collagen?

Answer 4

• Family of fibrous proteins found in all multi cellular organisms
• Major protein components of bone, tendon and skin and different collagen component encoded by 28 diff genes
• Each collagen molecule made up of three alpha chains and can be a homotrimer or a heterotrimer

Question 5

What are the different types of collagen?

Answer 5

1. Type 1 collagen has chains from two genes and heterotrimer with composition alpha [α1(I)]2 [α2(I)]
2. Type II and III collage are homotrimers having only one chain type and their compositions are [α1(II)]3 and [α1(III)]3

Question 6

Describe collagen

Answer 6

• The alpha chains form a triple helix
• In fibrillar collagen, each alpha chain is approx. 1000 amino acids long, forming a left handed helix
• Primary sequence of collagen proteins has glycine-x-y repeat where x is often proline and y is often hydroxyproline
• To form a stiff triple helical structure every third position in there must be occupied by the amino acids glycine, as only aa small enough to occupy interior
• Hydroxylation of purine is a post transitional modification which contributes to interchain hydrogen bond formation
• Lysine and hydroxylysine are similarly modified in formation of covalent cross linkages
• Crosslinking only happens after collagen secreted
• Vitamin C deficiency results in underhydroxylated collagens (leads to scurvy) due to enzymes Proly hydroxyls and Lysl hydroxylase rehiring vitamin C as a co-factor for functionality.

Question 7

Describe collage biosynthesis

Answer 7

1. Collagen biosynthesis and secretion follows normal pathway for a secreted protein
2. But collagen alpha chains synthesised as longer precursors called pro-alpha chains by ribosomes attached to ER
3. Pro-alpha chains undergo series of covalent modification and fold into triple-helical pro collagen molecules before their release from cells

Question 8

How is tensile strength achieved? What is a disease?

Answer 8

• Some collagens are fibril-associated and regulate the organisation of collagen fibrils in tissues
• Staggered arrays of tropocollagen molecules form fibrils which ultimately arrange to form collagen fibres
• Tensile strength is provided by the fibres being in parallel bindles, these rest tensile force in one direction
• Ehlers-Danlos syndromes (EDS) group of inherited connective tissue disorders (stretchy skin and loose joints). Arise mutations in collagen negatively affect collagen structure/production/processing

Question 9

What are non-fibril forming collagen?

Answer 9

• Not all collagens form fibrils, important non-fibrillar collagen is network forming collagen type IV, present in basement membranes
• Collagen type IV molecules associate laterally between triple-helical segments as well as head-to head and tsil-to tail between globular domains to give dimers, tetramers and higher order complexes

Question 10

What is a basement membrane and some diseases?

Answer 10

• Flexible thin mats of extracellular metric underlying epithelial sheets and tubes
• BM surround muscle, peripheral nerve and fat cells and underlie most epithelia
• Highly specialised extracellular matrices with distinct repertoire of collagens, glycoproteins and proteoglycans
• In kidney form key part of filtration unit as the glomerular basement membrane (GBM)
• In diabetic nephropathy: accumulation of EM lead to thickened BM - restricts renal filtration + lead to renal failure
• In Alport syndrome: mutations in collagen IV - abnormally split and laminated GB - associated progressive loss of kidney function + hearing loss

Question 11

Describe elastic fibres

Answer 11

• Elastic fibres for elasticity of tissues such as skin, blood vessels and lints
• Collagen and elastic fibres interwoven to limit the extent of stretching
• Core made up of protein elastin and microfibrils and fibrillin

Question 12

Describe fibrilin

Answer 12

• Integrity of elastic fibres depends upon mcirofibrils containing protein fibrillin
• Mutations in protein fibrillin-1 assoicated with Marfan’s syndrome (skeletal, ocular and cardiovascular system manifestations and can be predisposed to aortic ruptures)

Question 13

Describe elastin

Answer 13

• Hydrophobic regions and a-hlical regions rich in alanine and lysine alternate along polypeptide chain
• Many lysine chains are covalently cross-linked

Question 14

What is a modular architecture of ECM proteins?

Answer 14

• Most ECM proteins v large and composed of characteristic protein domains of 50-200 aa
• Multi functionality of ECM proteins result of modular structure
• Many large modular proteins are mutliadhesive, binding various matrix component and cell-surface receptors

Question 15

What are laminins?

Answer 15

• Are heterorotrimeric proteins made up of alpha chain, beta chain and a gamma chain which from cross shaped molecules
• V large proteins with each chain large molecular weight
• Multiadhesive proteins which can interact with a variety of CS receptors such as interns and dystroglycan
• Can self-associate as part of basement membrane matrix
• Can also interact with other matrix components such as type IV collage, nidogen and proteoglycans
• Mutations in specific chains are associated with inherited debases such as muscular dystrophy and epidermolysis bullosa
• Congenital muscular dystrophy can arise from an absence of alpha2 chain in laminin 2
• Symptoms include hypotonia (abnormally decreases muscle tension), a generalised weakness and deformities of the joints

Question 16

What are fibronectins?

Answer 16

• Family of closely related glycoproteins of ECM which also found in body fluids
• Can exist as an insoluble fibrillar matrix or as a soluble plasma protein
• Derived from single gene, with alternate splicing of mRNAs, giving rise to the different types
• Multi-adhesive proteins, made up of large multi domain molecule linked together by disulphide bonds
• Able to interact with CS receptors and other matrix molecules
• Play important roles in regulating cell adhesion and migration e..g. in embryogenesis and tissue repair
• Important for wound healing, helping to promote blood clotting
• Form a mechanical continuum with the actin cytoskeleton of many cell types
• Integrin receptors at the cell surface (CS) provide the linkage between the matrix and cytoskeleton

Question 17

What are the different proteoglycan families?

Answer 17

1. Basement membrane proteoglycans e.g. perlecan
2. Aggregating proteoglycans (interact with hyaluronan) e.g. aggrecan
3. Small leucine-rich proteoglycans e.g. decorin
4. Cell surface proteoglycans e.g. syndecans 1-4

Question 18

What are GAG chains?

Answer 18

• Made up of repeating disaccharide units with one of the two sugars being an amino sugar a suga (a sugar in which a hydroxyl group is replaced with an amine group)
• Many GAGs are sulphated or carboxylated and as a result carry a high negative charge
• This cadger attracts a cloud of cations including Na+ resulting in large amounts of water being sucked into the ECM

Question 19

What is cartilage?

Answer 19

• Cartilage has a matrix rich in collage with large quantities of GAGs trapped within the meshwork
• Balance of swelling pressure is negated by the tension in the collagen fibres, generating great tensile strength e.g cartilage lining knee joint (synovial cartilage) can support pressures in excess of hundred of kg/cm squared
• Small proteoglycans can have a single GAG chain attached and some large one carry up to 100 GAG chains

Question 20

What are the four main GAG groups?

Answer 20

1. Hyaluronan
2. Chondroitin sulfate and dermatan sulfate
3. Heparan sulfate
4. Keratan sulfate

Question 21

What is hyaluronan?

Answer 21

• Also called hydronic acid spun out directly from an enzyme embedded in the plasma membrane and all other GAGs are synthesised and attached to their core proteins in the endoplasmic reticulum and Golgi apparatus inside the cells
• It is found in ECM of soft connective tissues
• Distinct from other GAGs as it is simply a carb chain without core protein
• Unsulfated and made up of repeating disaccharides which can number up to 25,000 sugars
• Can undergo high degree of polymerisation
• Hyularonan chains can occupy relatively large volume
• Typically of high viscosity e.g. vitreous humour of eye and synovial fluid of joints and in latter location it plays key role in protecting cartilaginous surface from damage

Question 22

What is aggrecan?

Answer 22

• Major constituent of cartilage ECM
• The GAGs are highly sulphated increasing their negative charge
• Large numbers of carboxyl groups and these multiple negative charges attract cations such as Na+ that are osmotically active
• So large quantities of water being refined by highly negative charged environment
• Under compressive loads water is given up but regained once load is reduced so aggrecan in the cartilage matrix is perfectly suited to resist compressive forces

Question 23

What is osteoarthritis?

Answer 23

An erosive diseased resulting in excessive ECM degradation
The cushioning properties of cartilage over the end of bones are lost, with increasing age, aggrecan is cleaved by aggrecanases and metalloproteinases
This results in loss of aggrecan fragments to the synovial fluid

Question 24

What are fibrotic diseases?

Answer 24

Arise as a result of excessive production of fibrous connective tissue

Question 25

What are some diseases from laminin? What are the symptoms?

Answer 25

1. Mutations in specific chains are associated with inherited diseases such as muscular dystrophy and epidermolysis bullosa
2. Congenital muscular dystrophy can arise from an absence of the α2 chain in laminin 2
3. Symptoms include hypotonia (abnormally decreased muscle tension), a generalised weakness and deformities of the joints.

Question 26

What do fibronectins do and what are their roles?

Answer 26

1. Fibronectins are able to interact with cell surface receptors and other matrix molecules
2. They play important roles in regulating cell adhesion and migration in a variety of processes, notably embryogenesis and tissue repair.
3. They are also important for wound healing, helping to promote blood clotting (ALS2 - Haemostasis)
4. . Fibronectins form a mechanical continuum with the actin cytoskeleton of many cell types

Question 27

What are proteoglycans?

Answer 27

Core protein + one (or more) glycosaminoglycan chain

Question 28

Describe GAGs

Answer 28

-Long unbranched sugars consisting of repeating disaccharide units
-Occupy huge volume relative to mass
-Produce hydrated gels (resistant to compression)
-Sulfated and highly negatively charged
-Chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate
Hyaluronan is unique and has no core protein (simple carbohydrate chain)

Question 29

What is decorin (proteoglycans)?

Answer 29

1. Single GAG attached
2. .Dermatan sulfate chain
3. Binds to collagen fibers and regulates fibrillogenesis
4. Lack of results in fragile skin with reduced tensile strength

Question 30

What is aggrecan?

Answer 30

• major constituent of the cartilage extracellular matrix
• GAGs are highly sulfated, increasing their negative charge
• Also present are large numbers of negatively carboxyl groups. These multiple negative charges attract cations such as Na+ that are osmotically active. This in turn leads to large quantities of water being retained by the highly negatively charged environment.
• Under compressive load, water is given up, but regained once the load is reduced. Therefore, aggrecan in the cartilage matrix is perfectly suited to resist compressive forces.

Question 31

What happens in ECM degradation and excess?

Answer 31

Degradation- osteoarthritis

Excess ECM deposition- fibrotic lung, liver cirrhosis

Question 32

Describe collagen types

Answer 32

-Non-fibrillar collagens
1. Network forming collagen IV
2. Present in all basement membranes
-Collagen IV network
1. Can associate laterally and at the ends
-Hydroxyl group contributes to interchain H bond formation
-Lysine and hydroxylysine modified in formation of covalent cross-linkages
-Requires vit C and iron
Tensile and stability

Question 33

What is EDS?

Answer 33

• Ehlers–Danlos syndromes (EDS) are a group of inherited connective tissue disorders whose symptoms include stretchy skin and loose joints.
• Several of these can arise due to mutations in collagen, which negatively affect collagen production, collagen structure or collagen processing.

Question 34

What is elastin?

Answer 34

1. Core protein elastin, microfibrils rich in fibrillin

2. Mutation in fibrillin-1 leads to Marfan’s Syndrome