Laminin

Tuesday, Feb. 15

Group Three:

Claire Paulson, Andrew Kalas, Jacquie Fable & Chris McKay

Objectives

Predict what would occur in the absence of the basement membrane. (Level 2, Understand)

Compare and contrast, in both structure and function, Laminin with other important ECM proteins (Level 4, Analyze)

Design a potential treatment that could be applied to a patient with a laminin deficiency disease. (Level 6, Create)

Figure 19-43 Molecular Biology of the Cell (© Garland Science 2008)

Laminin in the ECM

Introduction to the Basal lamina Cell Signaling

Presents a microenvironment Tissue development, repair, normal homeostasis Errors can result in cancer, diabetes and

autoimmune complications Proteoglycans vs. glycoproteins?

Ratio of protein to carbohydrates present

Biological Structure of the Basal Lamina

The basement membrane is the earliest portion of the extracellular matrix formed during embryogenesis.

Provides signals to adjacent cells

 Force-driven signals originating between the basal lamina components and linked cell adhesion receptors (integrins) is communicated to the interior of cells through a mechanotransduction system to influence cell polarity, metabolism, fate, and migration.

Overall function of basal lamina The basal lamina is a layer

of the ECM that is secreted by epithelial and mesenchymal cells.

Found on the basal side of polarized epithelial cell sheets

separates them from the underlying connective tissue

Surround individual muscle cells, fat cells, and cells lining peripheral nerve cell axons (as Schwann cells)

Mainly serves as a base on which cells can grow

Figure 19-40 Molecular Biology of the Cell (© Garland Science 2008)

The basement membrane proteins laminin, collagen IV, nidogen/entactin, fibulin-1, perlecan and SPARC all bind integrins. Various integrins all bind to the regions indicated for the various BM components. The arrows indicate the sites at which the various BM proteins bind to each other.

Interactions with the Basal Lamina Binds integrins to signal to other cells

Loss of these signals leads to lethality of mouse embryos at the pre-implantation stage

Collagen (type IV) Insoluble Gives basal lamina strength

Nidogens (nidogen-1) Bind laminin to collagen Formation of the laminin/nidogen-1 complex would be a key event during basement membrane deposition andepithelial differentiation

Heparin Sulfate Binds ligands involved in metastasis and blood coagulation

Formation during Embryogenesis Polymerization of laminin and collagen IV in

two separate networks associated together by nidogen

The mechanism of basal lamina formation and insolubilisation of the different components into a basement membrane, in vivo, is unknown

Integrins are first and primary way to mediate cell-cell interaction

Compartmentalization of Tissue with the Basal Lamina

Separates connective tissue from the epithelia, nerves, and muscle tissue.

Controls interactions which influence cell polarity, differentiation and migration

(A) Mucosal melanosis of the lip. Numerous melanocytes above the basal lamina appear as a brown ribbon

(B) pseudostratified respiratory mucosa of the nose with predominantly ciliated cells. Goblet cells have clear cytoplasm. The basal cells (arrow) are lying on thin basal lamina

Distribution of proteoglycans

In vitro testing Amount of laminin, collagen,

nidogen, and heparin sulfate proteoglycans varies based on tissue

Cannot be isolated as a complete structure or reconstituted in vitro. Some components can be purified Isolated molecules lack the molecular

interactions that constitute basement membranes

Isoform composition of the four major basement membrane components varies with tissue type

Activity 1: Pick a tissue!

Muscl

e!

Kidne

y!Ski

n!

Brain

!

Activity 1 (10 min)

Predict what would happen in your tissue if the basal lamina was absent.

- Tissue structure- Boundaries- Abnormalities or benefits

Laminin’s Composition heterotrimeric

protein composed of various α, β, and γ subunits

most subunits contain similar LE (or EGF like) and LN domains

only the alpha subunit has LG domains

Structure

each subunit contains multiple 7 long amino acid sequences that enable the subunits to come together

the structure of the coiled coil maximizes the number of non-covalent bonds formed between the subunits and confers structural stability to the completed timer

once the coiled coil is formed it is covalently strengthened by disulfide bonds

Domains of the , , and γ chains LG domain can bind

various ECM molecules such as heparin, α dystroglycan , sulfatides, integrins, nidogen, and fibulin

LG domains are often found to be present in tandem which increases the efficiency of ligand binding

LN domains are primarily involved in binding to other laminins

Shapes different subunit types

create the diverse number of laminins (presently 18 are known)

not all the combinations of the three chains have been found to occur in living organisms

Some Biochemistry

LE Domain

-left, the classical look of disulfide connectivity based on X ray structures of the laminin γ1 chain (includes nidogen binding site)

-right, through mass spectometry they determined a different disulfide bonding patter of LE domains 1-4

this suggests that LE domains differing in function also differ in their disulfide patters

LG domain-Integrin Interactions the C terminus of the

β, and γ chains fine tunes the conformation of the LG domains so that they can interact with integrin

the dotted circle shows the integrin binding site

an aspartic acid residue found at the C terminus of the γ chain helps coordinate the divalent metal ion

Heterotrimer Formation

1) Glycosylation of subunits• Rough ER• stabilizes & protects

2) Association of and γ chains

3) chain combines with dimer• rate limiting step

4) Terminal glycosylation• Golgi

5) Trimer secreted from cell• Epithelial &

mesenchymal cells

6) Proteolytic processing

2

3

6

5So how do these laminin molecules form?

Discovery of Laminin Synthesis

the β and γ chains associate with one another first, followed by the α chain which combines with the dimeric complex

the α chains, the only chain which can be secreted free, drives secretion of paired βγ dimers

thus, the coiled coil domain of the long arms are crucial for assembly of the three chains of laminin

Polymerization Laminin-111 has been

the most thoroughly studied laminin and has helped understand how laminin contributes to the basement membrane architecture

it was determined that laminins interact with each other between the LN domains of the short arms thus forming a meshwork polymer

Receptor Mediated Laminin Assembly this model suggests that

laminin serves as an integrating receptor during its assembly into the basement membrane

by binding to integrin and dystroglycan, laminin can polymerize through short arm interactions and thus reorganize the receptors

this laminin/receptor network represents a widespread regulatory mechanism

Laminin Tissue Distribution 1 chain

embryogenesis: epithelial cells adult: liver, kidney, & reproductive organs

2 chain neuromuscular system

3 skin & epithelial cells

4 cells of mesenchymal origin vascular endothelial cells, cells derived from bone barrow

5 widely expressed epithelial, neuromuscular, vascular tissues embryogenesis

Laminin Processing

chains chains

γ chains

3A3

γ2

aka. Laminin 5 Skin, placental, mammary glands Hemidesmosomes, cellular migration

Processing of 3 chain unprocessed – migration processed – hemidesmosomes alters intergrin binding

Processing of γ2 chain DIII fragment binds erbB1 phosphorlyation of beta of 24

Marinkovich, 2007

Durbeej, 2010

Laminin Processing : Laminin-332

How is this possible?

!

Tzu & Markinkovich, 2008

Laminin-332 Where else is laminin-332

found? Cell migration

wound healing

What type of 3 chain?

Margadant & Sonnenberg, 2010

tumor invasion

Activity (min) Compare and contrast the major ECM proteins

we’ve learn about so far: collagen, fibronectin, & laminin

Collagen Fibronectin Laminin

Structure

Class of molecules

Processing

Connections within the ECM

Function

Importance in Embryogenesis

Location within the body

Associated diseases

Collagen Fibronectin Laminin

Structure

Class of molecules

Processing

Connections within the ECM

Function

Importance in Embryogenesis

Location within the body

Associated diseases

Trimeric triple helixGly-X-Y sequence

Trimeric, helical domain, globular ends, disulfide bonding

Dimer with modules I, II, III, disulfide bonding, sheets

Essential Essential -- Embryonic epithelia, placenta

Essential -- Blastocoele formation

proteins glycoproteinsglycoproteins

Pro- chain, glycosylation, Pro-collagen, N & C terminal cleaved collagen

Glycosylation, -γ dimer, addition chain, proteolytic cleavage

Dimers secreted

Integrins, MMPs, laminin

Collagen, integrins, dystroglycan, heparan sulfate proteinglycans

Cryptic sitesIntegrins, collagen, heparan , fibrin

Structure,/scaffolding signaling, movement, strength

Signaling, movement, tissue integrity, cell-matrix adhesion

Cell adhesion, cell growth, inhibition of apoptosis, movement, differentiation

ECM everywhere Basal lamina – separates epithelial from connective tissue

ECM everywhereBlood plasma

Chondroysplasias, osteoarthritis , osteoporosis, scurvy, lupus erythematosus

Muscular dystrophy, epidermolysis bullosa, cardiomyopathy, Pierson syndrome, cancer progression

Cancer progression

Laminin Mutations in Disease

Durbeej, 2010

Where is 2 chain expressed?

What kind of disease could result?

Laminin Mutations in Disease

Durbeej, 2010

What kind of disease could result? Where is 3 chain

expressed?

Laminin Mutations in Disease

Durbeej, 2010

Where is 4 chain expressed?

What kind of disease could result?

Laminin Mutations in Disease

Durbeej, 2010

Where were 1 and 5 chains important?

Why are there no diseases associated with loss of these chains?

Embroygenesis!

Cylindromatosis Rare genetic disorder

laminin-332 defective γ2 processing

Basement membrane thickens Multiple benign epithelial tumors

Tzu & Marinkovich, 2008

What was the γ2 involved in?

Muscular Dystrophy : MDC1A

LAMA2 gene mutation/deletion laminin-211 Where is this?

muscle, heart, peripheral nerve, testis skeletal muscle

dystrophin glycoprotein complex (DGC) and ECM

Phenotype: muscle weakness hypotonia joint stiffness inability to walk dysmyelinating peripheral neuropathy brain defects

Durbeej, 2010

no 2 chain C

Reed, 2009

Pierson syndrome Mutation in 2 chain gene

Glomerular defects

Ocular abnormalities

Muscular and neurological defects

Zenker, 2004

renal failure

loss of vision

early fatality

From Gene to Protein

Transcription

Translation

From LAM to Laminin

LAMA3 LAMC2LAMB3

LAMA3 mRNA LAMC3 mRNALAMB3 mRNA

Β3γ2 Complex

Laminin-332

Activity 3 (10 Min) You have a patient that is exhibiting

symptoms of a disease cause by a defect in correct Laminin expression

To determine a course of treatment, you must first determine the origin of the defect

Activity 3 (10 Min) Genetic defect in the gene itself Deletion of the gene itself Malfunction in RNA Polymerase Ribosomal Defect Defects in processing (subunit goes

unprocessed) Auto-Immune

Endometriosis: Auto-Immune Laminin Disease?

Laminin 111 and 511 critical in embryonic development

Inagaki et al. American Journal of Reproductive Immunology (2011). http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0897.2010.00956.x/abstract

Misplaced tissue causes creation of auto-antibodies

Auto-antibodies attack uterine tissue, causing infertility

Laminin-332 and the Epithelial Basement Membrane

LAMA3

LAMC2

LAMB3

Correct LAMB3 Expression

Incorrect LAMB3 Expression

Epidermolysis Bullosa (EB)

Severe skin disorder characterized by hemidesmosome dysfunction

Disconnect between Epidermis and Dermis

Characterized by severe blistering and loss of skin

Sawamura et al. Journal of Dermatology (2010); 37: 214-219

Junctional Epiermolysis Bullosa

Buchroithner et al. Laboratory Investigation (2004); 84: 1279-1288

Activity 4 (20 Minutes) You have a patient who is exhibiting blisters

on their skin and you suspect EB.

Explore ways to confirm JEB and the possible ways in which it could be treated.

Possible Answers Slide Diagnose

Electron Microscopy DNA Sequencing ELISA

Treatment Gene Therapy Stem Cells Artificial Grafts Artificial Protein Therapy

Objectives

Predict what would occur in the absence of the basement membrane. (Level 2, Understand)

Compare and contrast, in both structure and function, Laminin with other important ECM proteins (Level 4, Analyze)

Design a potential treatment that could be applied to a patient with a laminin deficiency disease. (Level 6, Create)

ReferencesDurbeej M (2010) Laminins. Cell Tissues Res. 339:

259-268.

Marinkovich MP (2007) Laminin 332 in squamous-cell carcinoma. Nature Reviews Cancer. 7: 370-380.

Reed UC (2009) Congenital muscular dystrophy Part I: a review of phenotypical and diagnostic aspects. Arq Neuropsiquiatr. 67(1): 144-168.

Tzu J & Marinkovich MP (2007) Bridging structure with function: Structural, regulatory, and developmental role of laminins. IJBCB. 40: 199-214.