Transcript Laminin presentation Tuesday

Laminin
Group Three:
Claire Paulson, Andrew Kalas, Jacquie Fable & Chris McKay
Tuesday, Feb. 15
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)
Laminin in the ECM
Figure 19-43 Molecular Biology of the Cell (© Garland Science 2008)
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


Collagen (type IV)



Loss of these signals leads to lethality of
mouse embryos at the pre-implantation stage
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 and
epithelial 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!
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 noncovalent 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
•
6
Rough ER
stabilizes & protects
So
how do these laminin molecules
Association of  and γ
5 form?
•
2)
3)
chains
 chain combines with
dimer
•
4)
Terminal glycosylation
•
5)
Golgi
Trimer secreted from
cell
•
6)
rate limiting step
Epithelial & mesenchymal
cells
Proteolytic processing
2
3
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



2 chain


skin & epithelial cells
4



neuromuscular system
3


embryogenesis: epithelial cells
adult: liver, kidney, & reproductive organs
cells of mesenchymal origin
vascular endothelial cells, cells derived from bone barrow
5



widely expressed
epithelial, neuromuscular, vascular tissues
embryogenesis
Laminin Processing
 chains
 chains
3
3A
γ chains
γ2
Laminin Processing : Laminin-332
!



aka. Laminin 5
Skin, placental, mammary glands
Hemidesmosomes, cellular migration

Processing
of 3
chain
How is this
possible?




unprocessed – migration
processed – hemidesmosomes
alters intergrin binding
Processing of γ2 chain


DIII fragment binds erbB1
phosphorlyation of beta of 24
Marinkovich, 2007
Durbeej, 2010
Tzu & Markinkovich, 2008
Laminin-332

Where else is laminin-332 found?

Cell migration

wound healing

What type of
3 chain?
tumor invasion
Margadant & Sonnenberg, 2010
Activity (min)

Compare and contrast the major ECM proteins we’ve
learn about so far: collagen, fibronectin, & laminin
Collagen
Structure
Class of molecules
Processing
Connections within the ECM
Function
Importance in Embryogenesis
Location within the body
Associated diseases
Fibronectin
Laminin
Collagen
Structure
Trimeric triple helix
Gly-X-Y sequence
Class of molecules
proteins
Processing
Pro- chain, glycosylation,
Pro-collagen,
N & C terminal cleaved 
collagen
Fibronectin
Dimer with modules I, II,
III, disulfide bonding,
 sheets
glycoproteins
Dimers secreted
Laminin
Trimeric, helical domain,
globular ends, disulfide
bonding
glycoproteins
Glycosylation, -γ
dimer, addition  chain,
proteolytic cleavage
Connections within the ECM
Integrins, MMPs,
laminin
Cryptic sites
Integrins, collagen,
heparan , fibrin
Collagen, integrins,
dystroglycan, heparan
sulfate proteinglycans
Function
Structure,/scaffolding
signaling, movement,
strength
Cell adhesion, cell
growth, inhibition of
apoptosis, movement,
differentiation
Signaling, movement,
tissue integrity, cellmatrix adhesion
Importance in Embryogenesis
Essential
Essential -- Blastocoele
formation
Essential -- Embryonic
epithelia, placenta
Location within the body
ECM everywhere
ECM everywhere
Blood plasma
Chondroysplasias,
osteoarthritis ,
osteoporosis, scurvy,
lupus erythematosus
Cancer progression
Associated diseases
Basal lamina – separates
epithelial from connective
tissue
Muscular dystrophy,
epidermolysis bullosa,
cardiomyopathy, Pierson
syndrome, cancer
progression
Laminin Mutations in Disease
What kind of disease could result?
Where is 2 chain expressed?
Durbeej, 2010
Laminin Mutations in Disease
What kind of disease could result?
Where is 3 chain expressed?
Durbeej, 2010
Laminin Mutations in Disease
What kind of disease could result?
Where is 4 chain expressed?
Durbeej, 2010
Laminin Mutations in Disease
Where were 1 and 5
chains important?
Embroygenesis!
Why are there no diseases
associated with loss of these
chains?
Durbeej, 2010
Cylindromatosis

Rare genetic disorder


laminin-332
defective γ2 processing
What was the γ2 involved in?
Tzu & Marinkovich, 2008


Basement membrane thickens
Multiple benign epithelial tumors
Muscular Dystrophy : MDC1A


LAMA2 gene mutation/deletion  laminin-211
Where is this?


muscle, heart, peripheral nerve, testis
skeletal muscle
no 2 chain C


dystrophin glycoprotein complex (DGC) and ECM
Phenotype:






muscle weakness
hypotonia
joint stiffness
inability to walk
dysmyelinating peripheral neuropathy
brain defects
Durbeej, 2010
Reed, 2009
Pierson syndrome

Mutation in 2 chain gene

Glomerular defects
 renal failure

Ocular abnormalities
 loss of vision

Muscular and neurological defects
 early fatality
Zenker, 2004
From Gene to Protein
Transcription
Translation
From LAM to Laminin
LAMA3
LAMB3
LAMC2
LAMA3 mRNA
LAMB3 mRNA
LAMC3 mRNA
Laminin-332
Β3γ2 Complex
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

Misplaced tissue causes creation of auto-antibodies

Auto-antibodies attack uterine tissue, causing infertility
Inagaki et al. American Journal of Reproductive Immunology (2011).
http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0897.2010.00956.x/abstract
Laminin-332 and the Epithelial Basement
Membrane
LAMB
3
Correct
Incorrect
LAMB3LAMB3
Expression LAMA
Expression
3
LAMC
2
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)
References
Durbeej 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.