Lecture 8 - Connective tissue disorders 2 Flashcards
Laminin: what is it, how abundant is it, and what is its structure?
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
Laminin 1: does it produce a network?
Its N-terminal globular domains promote polymerisation into a network with accessory proteins (nidogen) as well as other laminins in vitro
Underlying skin - the entire layer of laminin polymer
Laminin globular domains
LG1-LG5
LG1-3: integrins bind
LG4/5: dystroglycan heparin
Laminin network components
The laminin network is linked with the collagen IV network by accessory molecules:
- Nidogen
- Perlecan
- Integrin
Laminin genes: how many are there
5α, 3β, and 3γ chains and they associate to form 15 heterotrimeric combinations
There are more laminin isoforms than collagen isoforms
Laminin chains: α1 - where is it located and what phenotypes are presented when there is a deficiency of it?
Basement membranes of epithelial tissues during embryogenesis and some epithelial BMs in adults
Early embryonic fatality - only essential in embryonic tissue
Laminin chains: α2 - where is it located and what phenotypes are presented when there is a deficiency of it?
BMs of skeletal and cardiac muscles, peripheral nervous system, and central nervous system?
Severe congenital muscular dystrophy - lethal five weeks after birth
Laminin chains: α3 - where is it located and what phenotypes are presented when there is a deficiency of it?
Primarily in BMs of stratified epithelia
Lethal postnatal skin blistering - lethal three days after birth
Laminin chains: α4 - where is it located and what phenotypes are presented when there is a deficiency of it?
Vascular BMs
20% lethal, 80% are healthy and fertile
Laminin chains: α5 - where is it located and what phenotypes are presented when there is a deficiency of it?
Widespread
Lethal during midgestation
Laminin chains: β1 - where is it located and what phenotypes are presented when there is a deficiency of it?
Ubiquitous
Lethal E5.5 (?)
Laminin chains: β2 - where is it located and what phenotypes are presented when there is a deficiency of it?
Wide expression pattern
Lethal postnatally - defects with glomerular filtrate and NMJ
Laminin chains: β3 - where is it located and what phenotypes are presented when there is a deficiency of it?
Primarily in BMs of stratified epithelia
Lethal within 24 hours after birth
Laminin chains: γ1 - where is it located and what phenotypes are presented when there is a deficiency of it?
Ubiquitous
Lethal E5.5 (?)
Laminin chains: γ2 - where is it located and what phenotypes are presented when there is a deficiency of it?
Primarily in BMs of stratified epithelia
Lethal within 5 days after birth
Laminin chains: γ3 - where is it located and what phenotypes are presented when there is a deficiency of it?
Low expression in a few epithelial organs
N/A
Epidemolysis bullosa (EB):
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
Laminin 5: what chains are present, what does it do, and what collagens does it link to?
α₃β₃γ₂
Forms connections with integrins in hemidesmosomes and acts as a link to the underlying collagen
Collagen IV
Collagen VII: what is it and what does it do?
Anchoring fibres that link the BM to the collagen I network in the dermis - specific to basement membranes
Laminin 5 chain deficiencies: what conditions are caused, are they recessive or dominant, and what occurs in the sufferers?
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
Gene therapy for Laminin β3 chain mutations
Using retroviruses, a functional LAMβ3 gene can be given to patients own keratinocytes
Retroviruses: what are they and what are the potential problems using them
Allows you to incorporate a gene into the genome
Holoclones: what are they and what do they contain?
Holoclones are proliferative and contain stem cells
What are paraclones and meroclones?
Paraclones, meroclones - more differentiated
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?
- Structural links between the cytoskeleton and the matrix
Provides physical strength to tissues
Integrins
Integrins: what are they and what is their structure?
- Heterodimers of a and b subunits
- Large extracellular domain
- Single transmembrane spanning domain
- Short cytoplasmic domain (with one exception in b4)
RGD
Bind to fibrinectin (?) and (?)
Laminin receptors
b1
Collagen receptors
Leukocyte-specific receptors
Integrin connection to the cytoskeleton
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)
B4 integrin and skin integrity: how does b4 integrin relate to the skin and what occurs with b4 integrin deletion?
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)
Think - why should loss of the integrin be more severe than loss of Lm5?
Google me, im pre-housewives
PA-JEB: what is it, what occurs in those who suffer from it, and what are the symptoms?
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
EB simplex: how severe is it, what happens, how does healing work, what mutations are involved, and what other deletions are involved?
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)
Epidermolysis bullosa summary
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
The three key parts to tissue mechanical integrity
ECM, Cytoskeleton, and
