Lecture 8 - Connective tissue disorders 2

Question 1

Laminin: what is it, how abundant is it, and what is its structure?

Answer 1

Laminin is a high MW glycoprotein (around 800 kDa) with a cruciform structure seen in EM

After Col IV (which makes up about 50% of the BM), Laminin is the next most abundant BM protein

Cruciform structure:
* Three globular domains with spacers comprised of EGF repeats
* a β-chain and γ-chain that coil around the main central α-chain, forming a coiled long arm
* A chain C-terminal globular domain

Question 2

Laminin 1: does it produce a network?

Answer 2

Its N-terminal globular domains promote polymerisation into a network with accessory proteins (nidogen) as well as other laminins in vitro

Answer 3

Underlying skin - the entire layer of laminin polymer

Question 4

Laminin globular domains

Answer 4

LG1-LG5

LG1-3: integrins bind

LG4/5: dystroglycan heparin

Question 5

Laminin network components

Answer 5

The laminin network is linked with the collagen IV network by accessory molecules:

• Nidogen
• Perlecan
• Integrin

Question 6

Laminin genes: how many are there

Answer 6

5α, 3β, and 3γ chains and they associate to form 15 heterotrimeric combinations

There are more laminin isoforms than collagen isoforms

Question 7

Laminin chains: α1 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 7

Basement membranes of epithelial tissues during embryogenesis and some epithelial BMs in adults

Early embryonic fatality - only essential in embryonic tissue

Question 8

Laminin chains: α2 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 8

BMs of skeletal and cardiac muscles, peripheral nervous system, and central nervous system?

Severe congenital muscular dystrophy - lethal five weeks after birth

Question 9

Laminin chains: α3 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 9

Primarily in BMs of stratified epithelia

Lethal postnatal skin blistering - lethal three days after birth

Question 10

Laminin chains: α4 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 10

Vascular BMs

20% lethal, 80% are healthy and fertile

Question 11

Laminin chains: α5 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 11

Widespread

Lethal during midgestation

Question 12

Laminin chains: β1 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 12

Ubiquitous

Lethal E5.5 (?)

Question 13

Laminin chains: β2 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 13

Wide expression pattern

Lethal postnatally - defects with glomerular filtrate and NMJ

Question 14

Laminin chains: β3 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 14

Primarily in BMs of stratified epithelia

Lethal within 24 hours after birth

Question 15

Laminin chains: γ1 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 15

Ubiquitous

Lethal E5.5 (?)

Question 16

Laminin chains: γ2 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 16

Primarily in BMs of stratified epithelia

Lethal within 5 days after birth

Question 17

Laminin chains: γ3 - where is it located and what phenotypes are presented when there is a deficiency of it?

Answer 17

Low expression in a few epithelial organs

N/A

Question 18

Epidemolysis bullosa (EB):

Answer 18

Epidemolysis bullosa (EB):

Related group of conditions where skin blisters following mechanical trauma

Mutations affect the mechanical strength of the dermal/epidermal junction

Position of the break depends upon the genetic defect

The epidermal basement membrane is essential for mechanical strength of the skin

Cells are attached via the BM and anchoring fibres to the dermis

Any defect in the linkage from the dermis to the intermediate filament results in some type of EB

Novel approaches are becoming available to treat EB using gene therapy

Question 19

Laminin 5: what chains are present, what does it do, and what collagens does it link to?

Answer 19

α₃β₃γ₂

Forms connections with integrins in hemidesmosomes and acts as a link to the underlying collagen

Collagen IV

Question 20

Collagen VII: what is it and what does it do?

Answer 20

Anchoring fibres that link the BM to the collagen I network in the dermis - specific to basement membranes

Question 21

Laminin 5 chain deficiencies: what conditions are caused, are they recessive or dominant, and what occurs in the sufferers?

Answer 21

Leads to junctional EB (JEB) conditionals:
* Herlitz type JEB - lethal within the first few months after birth
* Non-Herlitz JEB - (?)

These are all autosomal recessive conditions

The epidermis has no chemical attachment to the dermis - it is lifted off
No difference at birth but blisters are developed postnatally and they die by day 3

Question 22

Gene therapy for Laminin β3 chain mutations

Answer 22

Using retroviruses, a functional LAMβ3 gene can be given to patients own keratinocytes

Question 23

Retroviruses: what are they and what are the potential problems using them

Answer 23

Allows you to incorporate a gene into the genome

Question 24

Holoclones: what are they and what do they contain?

Answer 24

Holoclones are proliferative and contain stem cells

Question 25

What are paraclones and meroclones?

Answer 25

Paraclones, meroclones - more differentiated

Question 26

Cell-ECM junctions in skin: what kind of links are present, what do these junctions do, and what is an example of an adhesion receptor?

Answer 26

• Structural links between the cytoskeleton and the matrix

Provides physical strength to tissues

Integrins

Question 27

Integrins: what are they and what is their structure?

Answer 27

• Heterodimers of a and b subunits
• Large extracellular domain
• Single transmembrane spanning domain
• Short cytoplasmic domain (with one exception in b4)

Question 28

RGD

Answer 28

Bind to fibrinectin (?) and (?)

Question 29

Laminin receptors

Answer 29

b1

Question 30

Collagen receptors

Question 31

Leukocyte-specific receptors

Question 32

Integrin connection to the cytoskeleton

Answer 32

The integrin cytoplasmic domain interacts with the cytoskeleton

Most integrins are associated with actin

In hemidesmosomes, they link to intermediate filaments made up of keratin (important for EB)

Question 33

B4 integrin and skin integrity: how does b4 integrin relate to the skin and what occurs with b4 integrin deletion?

Answer 33

Hemidesmosome-specific b4 integrins are required for the integrity of the skin

• Deletion of b4 integrins leads to the same phenotype as with laminin 5 deletions
• Loss of b4 integrin in mice results in the loss of hemidesmosomes but not the cell/cell adhesions that link to IF’s
• Similarly to the col IV and laminin deletions, loss of b4 also results in the absence of the a6 integrin subunit
• Cells are weakened internally, but the BM is intact
• Cells rip apart leaving part still attached to BM via the integrin but the formation of the BM appears to still occur (laminin 5 present)

Question 34

Think - why should loss of the integrin be more severe than loss of Lm5?

Answer 34

Google me, im pre-housewives

Question 35

PA-JEB: what is it, what occurs in those who suffer from it, and what are the symptoms?

Answer 35

Junctional epidermolysis bullosa with pyloric atresia (PA-JEB) is a rare autosomal recessive condition associated with the loss of a6b4 integrin

Neonatal mucocutaneous blistering and gastric outlet obstruction through loss of function in gastrointestinal, genitourinary and respiratory epithelium - this disease is fatal

Question 36

EB simplex: how severe is it, what happens, how does healing work, what mutations are involved, and what other deletions are involved?

Answer 36

EB simplex has a milder phenotype than other forms of EB - compared with JEB which has a 40% mortality in the first year

Usually restricted to blisters on regions subject to mechanical stress

Can heal without significant scarring

Mostly associated with mutations in keratins 5 and 14

Deletion of the b4 cytoplasmic tail in mice results in EB simplex (this phenotype will also be shown with mutations in any of the cytoplasmic proteins that link the b4 integrin cytoplasmic domain with intermediate filament)

Question 37

Epidermolysis bullosa summary

Answer 37

The epidermal basement membrane is essential for the mechanical strength of the skin

Cells are attached via the BM and anchoring fibres to the dermis

Any defect in the linkage from the dermis to the intermediate filament results in some type of EB

Novel approaches are becoming available to treat EB using gene therapy

Question 38

The three key parts to tissue mechanical integrity

Answer 38

ECM, Cytoskeleton, and