B3 biochem of the ECM

Question 1

ECM definition + roles

Answer 1

the complex network of secreted macromolecules located in the extracellular space, secreted by cellular components of the tissue (fibroblasts, chondrocytes, osteocytes, etc)

ROLES: maintenance of structure and cell support, nutrient distribution and growth, remodeling and removal of damaged tissue, angiogenesis

Question 2

Contents of the ECM (5)

Answer 2

1. Collagens
2. Elastin
3. Proteoglycans
4. GAGs
5. adhesive glycoproteins

!! fibers make up the matrix fibers, and the rest makes up the GROUND SUBSTANCE (soluble and hydrated easily)

Question 3

How many collagen types are there

Answer 3

28 (30% of total protein mass in the body and the most abundant component of ECM)

Question 4

Name some of the locations of the diff types of collagen

Answer 4

1 - skin, tendon, bone
2 - cartilage and vitreous body, intervertebral disc
3 - skin, muscle, vessels (usually with type 1)
4 - fetal tissue and placenta
5 - cartilage

Question 5

what are the specific characteristics of collagen molecules

Answer 5

-high tensile stiffness and strength
-high resistance to tension and pressure
-low resistance to compression
-different fibril diameter, packing and organisation

Question 6

Describe the collagen architecture (stages of polymer)

Answer 6

1. alpha chains (rich in amino acid residues, one in each three chains is glycine, and there is hydroxyproline and proline)
2. Tropocollagen: 3 alpha chains wrapped in a helical chain
3. Microfibrils: stragerred molecules of tropocollagen joined with covalent cross links
4. Fibrils: bundles of microfibrils
5. Collagen Fiber: bundles of fibrils

Question 7

Collagen synthesis process (8 steps)

Answer 7

INTRACELLULAR PHASE:
1. Protein synthesis of alpha chains on ribosomes of RER (since the protein is secretory) –> forms PREprocollagen
2. Hydroxylation of lysine in the RER (allows formation of cross links later)
3. Glycosylation of hydroxylysine starting in RER and completed in Golgi
4. Formation of S-S in extension peptides
5. Procollagen is formed (triple helix) in Golgi
6. Secretion of procollagen via trans Golgi out of cell

EXTRACELLULAR PHASE:

7. Cutting of the procollagen propeptides (N/C) domains to obtain tropocollagen
8. Self assembly of tropocollagen into microfibrils and fibrils - COLLAGEN MATURATION (cross links) and then assembly into fibers

Question 8

Collagen maturation definition

Answer 8

-The formation of covalent bonds between tropocollagen molecules when
-STAGGERED arrangement prevents degradation of collagen and increases strength

!!! This can only happen if there has been hydroxylation of lysine residues in the RER during collagen synthesis

Question 9

What is the key molecule needed for collagen synthesis and why

Answer 9

VITAMIN C:
-hydroxylation of proline uses dioxygenases which contain Fe and require OXYGEN
-one O in O2 is added to proline to form hydroxyproline, the other forms succinate
-During this process the Fe2+ is oxidised to Fe3+
-Vit C is needed to maintain the Fe in the reduced state so that the reaction can occur (Fe2+)

Question 10

Pathology arising from vit C deficiency

Answer 10

SCURVY
-non hydroxylated collagen chains unable to mature and stable helix isnt formed
-Loss of collagen in matrix causes vascular fragility, falling out of teeth and poor wound healing

Question 11

Describe the process of glycosylation of hydroxylisine

Answer 11

-attachment of galactose to the OH group of hydroxylysine (number of units depends on the type of collagen) to form a B-GLYCOSIDE BOND

-enzymes that mediate this: galactosyltransferases and glucosyltransferases

-donors of the groups: UDP-gal and UDP-Glc

Question 12

Describe the formation of S-S in extension peptides

Answer 12

-intrachain and interchain disulfide bonds formed in the Cterminal region of the propeptides

Necessary for the initiation of the triple helix to form (always from C end for correct alignment)

-intrachain S-S formed in the Nterminal

Question 13

How is procollagen secreted out of the cell

Answer 13

COPII- DEPENDENT PATHWAY (vesicle transport)

Question 14

What are the roles of procollagen propeptides

Answer 14

-N and C terminal which are cut off during tropocollagen formation (extracellular)

ROLES:
-correct alignment
-give higher ability to wind and coil

Question 15

What is collagen periodocity

Answer 15

Gaps created due to stagerred arrangement of tropocollagen molecules where electrons can pass through in a TEM. Creates alternating electron dense vs light regions

1 Gap = approx 40nm

Question 16

What are the types of cross links formed in collagen

Answer 16

ALL ARE COVALENT BONDS

1. INTRAmolecular –> lysine residues are deaminated and aldehydes formed. The aldol condesation od aldol groups is spontaneous and forms ALDOL cross links
2. INTERmolecular –> histidine-aldol cross links

Question 17

Final length of the collagen fiber

Answer 17

300 nm (around 1000 aa)

Question 18

Elastic fibers function

Answer 18

-provides flexibility to the tissue (eg for vessels, lungs, ligaments and skin)

Question 19

diff between collagen and elastin (6)

Answer 19

1. C is multigene, elastin is coded by 1 gene only
2. final length C = 1000 aa, E = 750 aa
3. locations found in and functions (C = tension and strength, E for flexibility)
4. aa composition: C = glycine and proline, E = 1/7 aa is valine and hence more hydrophobic
5. shape: C = triple helix, E = random comformation
6. differences in synthesis: C has extension peptides and makes aldol cross links, E has no extension peptdies and forms desmosine cross links

Question 20

SYNTHESIS of elastin process (steps)

Answer 20

1. ELN gene transcription in nucleus to make tropoelastin
2. tropoelastin interaction in ER with elastin binding proteins to fold
3. transport of complex to golgi
4. deposition of tropoelastin onto micofibril scaffold
5. tropoelastin desmosine cross link formation via LOX (LYSIL OXIDASE)

!! elastin (core protein) + microfibril scaffold sheath (made of fibrilin) = elastic fibers

Question 21

structure of proteoglycans

Answer 21

Core protein (hyaluronan) covalently bonded to GAG chains (negative linear polysacharides of repeating disacharide units)

Question 22

What is the units present in GAGs

Answer 22

linear polysaccharides of repeating disacharide units:

1. one is a uronic acid (GlcA or iduronic acid)
2. other is an amino sugar (GlcNAc/ GalNAc)

!! diff types have diff unit compositions + sulfation to increase -ve charge

Question 23

desribe the synthesis of GAGs

Answer 23

OCCURS IN ER MEMBRANE:
Core protein (Xyl-Gal-Gal) made in RER, and then addition of further groups is achieved using enzymes (glycosyltransferases and epimerases)

-sulfation can occur to the OH groups to increase the negative charges

!! proteoglycans are then made in the golgi

Question 24

specific characteristics of hyaluronic acid

Answer 24

-ubiquitous
-attracts water molecules well bcos its highly polar
-ROLE in wound healing and lubrication of synovial joints

!! DOESNT RECQUIRE ADDITIONAL SULFATION, AND IS MADE ON CELL MEMBRANE INSTEAD OF RER

Question 25

functions of proteoglycans

Answer 25

-very varible bcos they can have diff GAGs bound to surface (hence many functions)

-water absorption, collagen binding, cell migration and receptor activator for biomolecular signaling

Question 26

Collagen defects (clinical point)

Answer 26

1. EDS (hypermobility and elastic skin)
2. osteogenesis imperfecta (fragile bones)