Lecture 5: Connective Tissue (2): Laminin and Integrins

Laminin

• a high molecular weight glycoprotein (~800 kDa) with a cruciform (cross-like) structure:

  • 3 short arms with globular domains

  • 1 long arm with a globular domain at the end.

• It was originally isolated from a tumour in rodents and is primarily found in the basement membrane (BM).

• It is the second most abundant BM protein after ColIV

Structure of Laminin

• Made up of 3 separate polypeptide chains (alpha, beta and gamma) assembled into a cruicform structure

• Each of the chains is made up of an alpha helix that wraps around each other in a coiled-coil to form the long arm

  • Differs from the collagen-coiled-coiled domain

• N-termini form globular domains separated by EGF repeated

• C-Termini - alpha chain present and forms the C-terminal globular domain

  • Provides both structural and functional roles.

Formation of Laminin Network

• Laminin 1 spontaneously forms a network in vitro

  • Self assembles at high concentration

• The N-terminal globular domains promote and drives the polymerisation into a network by interacting with other laminins

  • Nidogen/ intactins interact with the domains and may have a role in stabilising the network

Formation of Laminin Network - a Chain

• The globular domain consists of 5 Lamin Globule domains that interact with cell surface receptors      

• 1-5 LG domains – together provide a binding site for different cell surface receptors e.g. integrins

  • C-terminal globular domain of the chain plays a role in binding

• The interaction of integrin with LG domains gives rise to a 3D structure that will assemble into a network

Integrins

• Cell surface glycoproteins that are receptors for various ECM components

  • Those that recognise laminin will bind to the LG domains

Organisation of Laminin Network

• Type IV collagen forms a 2D network through non-collagenous domains at its N and C termini, creating a structural backbone.

• Laminin forms its own network, integrated into the collagen network.

• Accessory proteins join the two networks, with collagen providing the structural backbone and laminin providing binding sites.

Laminin Genes

• 11 genes are present, but these only associate to form 15 different heterotrimeric combinations → more than for ColIV

  • 5 a-chains

  • 3 B-chains

  • 3 y-chains

    • Separate gene for each

• More variability present, with more tissue-specific expression and basement membranes having different types of laminin

  • mutation in chains gives rise to disease

Tissue-Specific Laminin Isoform: a1 LM chain

• BM of epithelial tissues during embryogenesis and some epithelial of BM in the adult

• Deficient phenotypes – early embryonic lethality

Tissue-Specific Laminin Isoform: B1 LM Chain

• Chain ubiquitously expressed

  • Deficient phenotype – lethal E5.5

Tissue-Specific Laminin Isoform: y1 LM Chain

• Chain ubiquitously expressed

  • Deficient phenotype – lethal E5.5

      a1,B1,y1 chain common in embryo – don’t develop past BM formation

a1B1y1 Laminin KO Mouse

• Mice indicate that some laminins are essential throughout the animal

  • The laminin is found in the embryonic BM

Tissue-Specific Laminin Isoform: a2 LM Chain

• Chain expressed in the BM of PNS, skeletal and cardiac muscle

  • deficient phenotype = severe congenital MD; lethal

Tissue-Specific Laminin Isoform: a3 LM Chains

• Primary chain expressed in the BM of stratified epithelia

  • Deficient phenotype – lethal postnatal skin blistering; death after 3

Tissue-Specific Laminin Isoform: B3 LM Chains

• Chain shows a wide expression pattern

  • Deficient phenotype – postnatally lethal – defects in glomerular filtration and NMJ

Pierson Syndrome

• Congenital nephrotic syndrome (glomeruli don’t filter properly) progressing to end-stage renal disease

• Simillar to ColIV in Alport’s

• Rare and lethal condition due to mutation in B2 – frameshift mutation in chains – non-functional laminin

• Laminin trimers that require B2 don’t form

Laminin

• (a5,B2,y1) and is expressed in the GBM, eye and synaptic BM, explaining the disease phenotype      

  • Eye abnormality      

  • Severe muscular hypotonia  

• Patients have a mutation in B2 – tissues that express the laminin trimer are implicated

How does a deficiency in Laminin B2 gene in mice phenocopy Pierson Syndrome?

• Laminin B2 is found in the GBM and laminin α1 in Bowman's capsule.

• B2 deficiency leads to replacement with the wrong isoform (β1 chain expression in the GBM).

• The incorrect laminin isoform can't form an effective filtration barrier, resulting in:

  • Proteinuria

  • Abnormal retinal and neuromuscular junctions

• Collagen IV remains unaffected but the wrong laminin isoform disrupts the function of both laminin and collagen IV, highlighting the need for both to function properly in filtration.

Epidermis

• It is attached to the dermis via a basement membrane (BM), providing mechanical strength.

• The average adult has 2 m² of skin weighing about 3.5 kg.

• Laminin isoforms in the epidermis bind to the dermis through laminin-integrin-collagen interactions.

• It consists of dead cells on top, with a living layer underneath (stratified epithelium).

• The boundary between the epidermis and dermis is the basement membrane (BM), linking the epithelium to the dermis' extracellular matrix (ECM).

Epidermis Bullosa (EB)

• A group of related conditions where skin blisters following mechanical trauma

  • The range of severity in this condition depends on where the mutation has occurred in the molecular connection

• Mutations affect the mechanical strength of the dermal/ epidermal junction

• Postion of the break depends upon the genetic defect

Laminin 5

• A skin-specific laminin found in the basement membrane (BM).

• It inks integrins on the epidermal layer through Collagen IV (α1α1α2).

• Collagen IV connects to Collagen VII, which extends out of the BM and links to Collagen I fibres (fibrillar collagens) in the dermis.

• This structure helps anchor and hold the skin layers together.

Hemidesmsomes

• Where cell surface receptors cluster and attach to the cytoskeleton

  • They are adhesion structures

Collagen VII

• Anchoring fibres that link the collagen network in the dermis

  • It is specific to the BM

Junctional Epidermis Bullosa

• An autosomal recessive disorder caused by mutations in Laminin 5 (a3B3y2)

  • Genes implicated LAMA3, LAMB3 LAMC2

• There are two types

  • Herlitz type

  • Non-Herlitz type

• Has a 40% mortality rate in the first year

Herlitz Type JEB

• Complete loss of any Laminin 5 hains in the epidermis

• It is lethal in the first few months after birth as the skin has no mechanical strength → lifts off the dermis

• An autosomal recessive condition

Non-Herlitz Type JEB

• A milder form of the condition caused by missense or splice site mutations → reduced Laminin 5 expression or partial function.

• An autosomal recessive condition

Use of Genetic Mouse Models to Show JEB

• Proves that the loss of laminin 5 causes junctional EB

• Laminin 5 gene deletion in mice phenocopies Herlitz JEB

  • No difference at birth but develop blisters post-natally

  • Mice die by day 3 as the skin detaches and there is no longer a water barrier present resulting in severe dehydration

Use of Genetic Mouse Models to Show Herlitz Type JEB

• Complete loss of laminin five chains due to a major gene deletion, rearing or chain termination mutation

• Results in a complete loss of function → fatal

Use of Genetic Mouse Models to Show Non-Herlitz Type JEB

• Reduced expression of laminin 5 chains due to mutations in the splice site thaWt reduce the efficiency of transcription splicing to make mRNA

  • It is a reduced loss of function mutation

• Mice can survive but have severe blistering

Why is JEB Suitable for Gene Therapy

• Due to the relatively easy accessibility of the skin

Case Study - Gene Therapy For Laminin B3 Chain Mutation

• Case: seven-year-old with a splice site mutation in exon 14 of LAMB3 (non-herlitz JEB)

  • Suffered from severe blistering since birth

  • Presented at a hospital following S. aureus infection, leading to loss of ~60% of epidermis

• Treatment: skin is taken and grown as a skin graft

  • Epidermal cells cultured

  • Retrovirus used to deliver functional B3 gene to keratinocytes

  • Cells grown into a sheet of transgenic skin to be grafted

• Outcome: After several months, the skin was repopulated by holoclones

  • Skin almost entirely derived from holoclones after 8 mon

Holoclones

• A stem cells containing a region that has proliferated

  • i.e they are proliferative and contain stem cells

• Paraclones and meroclones are more differentiated

Cell-ECM Junctions

• A continuous linkage between the cytoskeleton and the ECM fibres

• They are a mechanical connection through the plasma membrane.

  • Acts as structural links between the cytoskeleton and the matrix, providing physical strength to tissues.

Structure of Integrins

• They are cell-ECM adhesion receptors present on most cells

• Its heterodimers are composed of a and b- subunits that form the binding site

  • The combination of a- and B-subunit determines what ECM proteins the heterodimers are to bind

• It has a single transmembrane spanning domain → has ‘long legs’ that go through the plasma membrane

• Has a short cytoplasmic domain (with one exception in b4)

  • Each will go through the plasma membrane with a single alpha helix → short region that will connect to the cytoskelton

αβ Heterodimers

• Many different α and β subunits form numerous αβ heterodimers.

  • RGD receptors: Interact with ECM glycoproteins like fibronectin.

  • Collagen receptors

  • Leukocyte-specific receptors

  • Laminin receptors

• Have distinct but overlapping specificity for different ECM

Laminin Receptor

• a3, a6, a7 associated with laminin

• a6 can associate with B1 or B4 subunits

  • a6, B1 in MD (muscular dystrophy)

  • a6, B4 in EB (epidermolysis bullosa)

• These receptors have distinct but overlapping specificity for different ECM components.

Role of Integrin and ItS Cytoplasmic Domain

• Interacts with the cytoskeleton

• Assicates with the actin-myosin component of the cytoskeleton

Integrins B4

• Subunit interacts with hemidesmosomes linked to intermediate filaments made up of keratin (important for EB)

• Keratin fibres provide mechanical resistance; α6β4 integrin plays a central role in linking keratin fibres

Tissue-Specific Distribution of B1 Integrin Isoforms

• Expressed in early embryos and is widespread in tissues

  • Phenotype deficiency → low embryonic development and is lethal

Tissue-Specific Distribution of B2 Integrin Isoforms

• It is blood specific

Tissue-Specific Distribution of B4 Integrin Isoforms

• Phenotype deficiency is fatal, resulting in severe skin blistering

  • Skin detaches - similar phenotype to that of Laminin 5 deletion

• Loss of integrin-laminin connection resulting in the phenotype of severe blistering and skin detachmentnt

Hemidesomosme-Specific B4 Integrin

• They are required for the integrity of the skin

Loss of B4 Integrin In KO Mice

• Loss of hemidesmosomes, but not the cell/cell adhesions that link to Intermediate Fillaments’s      

• Cell-cell junctions are present due to different adhesion receptors

  • As a result, cells attach to each other but not to the Basement Membrane → Skin falls off

• Gives the same phenotype as laminin KO mice

Junctional Epidermis Bullosia With Pyloric Atresia (PA-JEB)

• A rare autosomal recessive condition associated with a loss of a6B4 integrin

• Results in neonatal mucocutaneous blistering and gastric outlet obstruction through loss of function in gastrointestinal, genitourinary and respiratory epithelium. 

• The disease is fatal – associated with non-functional B4 integrins

  • 2 distinct mutations (maternal/paternal) in the B4 allele

PA-JEB: Paternal Mutation in Integrin B4 Allele

• It consists of a one-base pair deletion causing a shift in the open reading frame, creating a downstream premature termination codon.

PA-JEB: Maternal Mutation in Integrin B4 Allele

• Occurs in a donor splice site and results in in−frame exon skipping involving the cytoplasmic domain of the polypeptide.

How May the Integrin a6 Subunit Become Absent

• Occurs following the loss of the B4 subunit:

  • Deletion of b4 integrin gene results in a loss of a6 integrin expression

• However, the basement still forms as seen by the presence of laminin 5, but is unable to attach to cells

Tissue Specific Distribution

• a5 present in early embryogenesis

• a6 lead to tissue-specific effects like skin blistering

Deletion of Integrin a6 Subunit

• It has the same effect as the loss of B4 in mice and humans

  • A missense mutation in α6 leads to the rapid degradation of the integrin

  • It can also cause loss of expression through premature stop codons.      

• Any genes associated with physically connecting the skin together lead to EB

EB Simplex

• A form of EB with a milder phenotype

• It is usually restricted to blisters on regions subject to mechanical stress

  • Localised blistering; skin is weaker but has some mechanical stability

• Heal without significant scarring

• Associated with mutations in keratin 5 and 14 → mutations inside the cell that affect the cytoskeleton’s connection to integrins

  • Mechanical integrity inside the cell is weakened due to mutations in the keratin (receptor and BM are still present)

Integrin B3

• Integrin subunit with a different cytoplasmic domain that is specialised to attach to the desmosomes

• It is linked to keratins function

Role of Integrin B4 in EB Simplex

• EB simplex is caused by mutations affecting integrin B4 subunit or its cytoplasmic linkers, leading to epidermis tearing from the basal membrane (BM).

• Mechanism:

  • Cells are weakened internally, but BM remains intact.

  • Partial loss of B4 integrin (e.g., loss of cytoplasmic domain) leaves part of the integrin attached to the BM.

  • Weakened ECM due to B4's role in connecting to the ECM.

  • Tearing of the epidermis occurs when integrins stay attached to the BM, but cells rip off → blistering seen

• Some mechanical strength is seen

ITGB4 Mutation

• A Heterozygous 2 bp deletion that leads to exon skipping and a 50 aa deletion in the B4 cytoplasmic domain where integrin binds to the IF cytoskeleoton

  • Mutations in any of the cytoplasmic components of the hemidesmosomes and the intermediate filaments can result in the disease phenotype

Mutation in EB Simplex

• Caused by mutation in the B4 integrin cytoplasmic domain

• Mutations in any of the cytoplasmic components of the hemidesmosomes and the IF that can result in the disease phenotype

Phenotype Differences in EB Simpelx

• The specific difference depends upon where the mutation in the mechanical linage has occurred

Role of the epidermal basement membrane in skin strength and its relation to EB

• The epidermal basement membrane (BM) provides mechanical strength for the skin by attaching cells to the dermis via anchoring fibres.

• Defects in the linkage from the dermis to intermediate filaments cause different types of epidermolysis bullosa (EB).

• Novel gene therapy approaches are being developed for EB treatment.

• Dystrophic EB is caused by mutations in Collagen VII.

Key Features of Collagen

• Collagenous domain made up of a tightly wound triple helix of the 3 collagen subunits

• Triple helix formation driven through the Gly-X-Y repeats that predominantly contain Pro and hydroxyprolines

• The combinations of the flexibility of glycine and the rigidity of the profile and hydroxyproline give rise to the tight triple helix formation

  • Characteristic of these proteins

Key Features of Laminin

• Composed of a triple helical structure based on each of the laminin subunits

• Long arm laminin subunits made up of alpha-helical structure

  • Alpha helix has a characteristic polypeptide structure  - 0.54 nm repeating structure of amino acids

  • 7 amino acids within 1 turn of the alpha helix

• Each of the long arms of the laminin subunits takes up this characteristic protein fold

Formation of Laminin Coiled Coiled Trimer

• Repeated sequence of 7 amino acids in the long arms of each chain allows the 3 subunits to form a coiled-coil trimer.

• This maximises non-covalent bonding between the chains.

• Amino acid sequence alternates between hydrophobic, hydrophilic, positive, and negative charges in each of the long arms.

• Alpha helical chains form hydrogen bonds and hydrophobic interactions, stabilising the trimer → forms the coiled-coil domain of the long arm

  • 3 alpha laminin alpha-helical are able to wrap around each other due to the alpha-helical repeat property

• At the C-terminal ends, disulfide cross-linkages stabilise the structure.

• This coiled-coil structure is distinct from collagen.

Requirements for Gene Therapy

• Approaches require a method for delivering the functional genes or repairing the defective gene in the tissues/ cells of the disease manifestation

  • This is made possible due to the life cycle of retrovirus, which allows them th. be use d in the delivery of functional copies of mutated genes

Retrovirus

• An RNA virus (small with a capsid and an envelope with proteins that allow them to bind to cells they will infect)

• They encode the enzyme reverse transcriptase which copies the viral RNA genetic material into a DNA copy to then form a double-stranded DNA helix which integrates into a host cell genome

  • Host cell has a permanent copy of the retroviruses genome integrated within, which is replicated each time the host cell divides

Wild Type Retroviruses

• They are able to replicate and make more RNA copies of the virus to form viral proteins to produce more infectious particles that are then secreted

• In GT they can be modified so that this function is disabled and that they no longer make more virus and only infect the host cell

  • Retrovirus can then be used to integrate a functional copy of a gene into a cell

Biopsy from A Patient With Low Laminin B3 Expression (and then following GT)

• Severe skin blistering

• Use of GT and a skin graft, the individual will have normal skin with regions of blistering

  • Biopsy then shows normal laminin expression → BM repaired