The Extracellular Matrix
Jeff Miner
7717 Wohl Clinic
362-8235
minerj@wustl.edu
Suggested Reading
**Overview: Lodish, Molecular Cell Biology (2008), Chapter 19, pp 801-841.
Lecture 1: The Extracellular Matrix
**Review: A Aszodi, KR Legate, I Nakchbandi, and R. Fassler: What mouse mutants teach us about
extracellular matrix function. Annu. Rev. Cell Dev. Biol. 22:591-621, 2006.
**Review: JF Bateman, RP Boot-Handford, SR Lamandé: Genetic diseases of connective tissues:
cellular and extracellular effets of ECM mutations. Nat. Rev. Genet. 10: 173-183, 2009.
KK McKee, D Harrison, S Capizzi, and PD Yurchenco: Role of laminin terminal globular domains in
basement membrane assembly. J. Biol. Chem. 282:21437-21447, 2007.
G Ge and DS Greenspan: BMP1 controls TGFβ activation via cleavage of latent TGF-binding
protein. J. Cell Biol. 175:111-120, 2006.
Lecture 2: Cell-Matrix Interactions
**Review: B-H Luo, CV Carman, and TA Springer: Structural basis of integrin regulation and
signaling. Annu. Rev. Immunol. 25:619-647, 2007.
**Review: KR Legate, E Montanez, O Kudlacek, and Reinhard Fassler: ILK, PINCH, and parvin: the
tIPP of integrin signalling. Nat. Rev. Mol. Cell Biol. 7:20-31, 2006.
Review: Y Mao and JE Schwarzbauer: Fibronectin fibrillogenesis, a cell-mediated matrix assembly
process. Matrix Biol. 24:389-399, 2005.
**Review: R Barresi and KP Campbell: Dystroglycan: from biosynthesis to pathogenesis of human
disease. J. Cell Sci. 119:199-207, 2006.
“Half of the secrets of the cell
are outside the cell.”
Dr. Mina Bissell
Oct. 17, 2007
Erlanger Auditorium
Basement Membrane Proteins Regulate
Mammary Cell Gene Expression:
Streuli et al,
J. Cell Biol. 1991
General Organization of Tissues
There are
Generic Tissue Structure
Stratified,
pseudostratified,
or monolayer
(aka stroma)
“Tube within a Tube” concept
“Tube Within a Tube”
Pancreas
A Compartmentalized Tissue
Even Hydra
“Mesoglea” (BM-like)
separates ectoderm from
endoderm
Shimizu, H. et al. Development 2002;129:1521-1532
Why do all multicellular animals have ECM?
• Act as structural support to maintain cell organization and
integrity (epithelial tubes; mucosal lining of gut; skeletal muscle
fiber integrity)
• Compartmentalize tissues (pancreas: islets vs. exocrine
component; skin: epidermis vs. dermis)
• Provide hardness to bone and teeth (collagen fibrils become
mineralized)
• Present information to adjacent cells:
– Inherent signals (e.g., RGD motif in fibronectin)
– Bound signals (BMP7, TGF, FGF, SHH)
• Serve as a highway for cell migration during development
(neural crest migration), in normal tissue maintenance (intestinal
mucosa), and in injury or disease (wound healing; cancer)
Types of ECMs
• Basement membrane (basal lamina)
– Epithelia, endothelia, muscle, fat, nerves
• Elastic fibers
– Skin, lung, large blood vessels
• Stromal or interstitial matrix
• Bone, tooth, and cartilage
• Tendon and ligament
Cells Need Receptors to Recognize
and Respond to ECM
•
•
•
•
•
Integrins
Dystroglycan
Syndecans
Muscle-Specific kinase (MuSK)
Others
Types of ECM Components
• Collagens
• Proteoglycans
– Perlecan, aggrecan, agrin, collagen XVIII
• Hyaluronan (no protein core)
• Large Glycoproteins
– Laminins, nidogens, fibronectin, vitronectin
• Fibrillins, elastin, LTBPs, MAGPs, fibulins
• “Matricellular” Proteins
– SPARC, Thrombospondins, Osteopontin,
tenascins
Generalizations
• Most ECM proteins are large, modular,
multidomain glycosylated or glycanated
proteins
• Some domains recur in
different ECM proteins
–
–
–
–
–
Fibronectin type III repeats
Immunoglobulin repeats
EGF-like repeats
Laminin Globular (G) domain
von Willebrand factor
Perlecan
Basement Membranes
• Specialized layers of extracellular matrix
surrounding or adjacent to all epithelia,
endothelia, peripheral nerves, muscle cells,
and fat cells
• Originally defined by electron microscopy as
ribbon-like extracellular structures beneath
epithelial cells
Basement Membrane
M. Loots, Univ. of Pretoria, S.A.
J. Schwarzbauer, Curr. Biol. 1999
Lamina Densa + Lamina Lucidae
Kidney Glomerular Basement Membrane
Fredrik Skarstedt and Carrie Phillips
Deep-Etch Electron Microscopy
Basement Membranes
• In general, basement membranes appear
very similar to each other by EM.
• But all are not alike!
• There is a wealth of molecular and
functional heterogeneity among basement
membranes, due primarily to isoform
variations of basement membrane
components.
Kidney Basement Membranes
Laminin 1
Laminin 2
Basement Membranes are Involved
in a Multitude of Biological Processes
• Influence cell proliferation, differentiation, and
migration
• Maintain cell polarization and organization, as well as
tissue structure
• Act as a filtration barrier in the kidney between the
vasculature and the urinary space
• Separate epithelia from the underlying
stroma/mesenchyme/interstitium, which contains a
non-basement membrane matrix
Primary Components of
All Basement Membranes
•
•
•
•
Collagen IV 6 chains form α chain heterotrimers
Laminin 12 chains form several α-β-γ heterotrimers
Entactin/Nidogen 2 isoforms
Sulfated proteoglycans Perlecan and Agrin are the
major ones; Collagen XVIII is another
History: The Engelbreth-Holm-Swarm (EHS) tumor:
A blessing with a caveat.
Laminin
Heterotrimers are composed of
one  , one , and one chain.
• 400 to 800 kDa cruciform, Y, or rodshaped macromolecules.
• Major glycoprotein of basement
membranes—it’s required!
• Chains are evolutionarily related.
• 5 alpha, 4 beta, and 3 gamma chains are
known. They assemble with each other
non-randomly.
• 15 heterotrimers described to date.
LM-521
Laminin
• All laminin chains share
structural homology
• Contain globular, rod (EGF-like
repeats), and coiled-coil
domains
• Alpha chains are unique,
contain a C-terminal laminin
globular “LG” domain, ~100
kDa
(New nomenclature)
The Laminin Trimers
Miner and
Yurchenco,
2004
Laminin Trimers Polymerize
• Laminin chains assemble into
trimers in the ER and are
secreted as trimers into the
extracellular space.
• Full-sized laminin trimers can
self-polymerize into a
macromolecular network
through short arm-short arm
interactions.
• The  chain LG domain is left
free for interactions with
cellular receptors.
Receptor-mediated Assembly
Involves LG domains and receptors on the surface of cells.
Results in laminin polymerization and signal transduction.
Laminin Mutations in Mice (M) and
Humans (H) Have Consequences
Lama1, Lamb1, Lamc1: Peri-implantation lethality (M)
Lama2: Congenital muscular dystrophy (M, H)
Lama3, Lamb3, Lamc2: Junctional epidermolysis bullosa (skin blistering)
(M, H)
Lama4: Mild bleeding disorder, moto-nerve terminal defects (M); cardiac
and endothelial defects (H)
Lama5: Neural tube closure, placenta, digit septation, lung, kidney, tooth,
salivary gland defects (M)
Lamb2: Neuromuscular junction and kidney filtration defects (M); Iris
muscle, neuromuscular, kidney filtration defects (H; Pierson syndrome)
Lamc3: Brain malformations, autism spectrum disorder? (H)
Sulfated Proteoglycans
•
•
•
•
Have protein cores with large
glycosaminoglycan (GAG) side chains
(from 1 to >100) attached to serines
Some PGs contain heparan sulfate
– Perlecan, Agrin, Collagen XVIII
(endostatin)
Others contain chondroitin, keratan or
dermatan sulfate
GAG chains are responsible for most of
the biological properties of
proteoglycans and provide charge to
basement membranes
Heparan sulfate:
Composed of D-glucuronate-2-sulfate +
N-sulfo-D-glucosamine-6-sulfate
Some Major Proteoglycan Family Members
From: Iozzo, R.V. (1998) Ann. Rev. Biochem. 67:609
From: Iozzo, R.V. (2001) J. Clinic. Invest. 108:165
Perlecan
• Found widely in basement membranes and in
cartilage.
• Contains domains similar to LDL receptor, laminin,
and N-CAM
• Binds to Collagen IV and to Entactin/Nidogen
Endorepellin: Domain V of Perlecan
• Exhibits anti-angiogenic activity
• Targets tumor vasculature
Endostatin: Noncollagenous Tail
of Collagen XVIII
• Exhibits anti-angiogenic activity
• Targets tumor vasculature
Type IV Collagen NC1 Domains
• Exhibit anti-angiogenic
activity
• Target tumor vasculature
Proteases Release Anti-Cancer Peptides
Laminin cleavages
MMP = Matrix Metalloproteinase
MT-MMP = Membrane Tethered MMP
From Zent and Pozzi, 2005
Agrin
• A HSPG found widely in basement membranes
• A modular protein containing domains homologous to
follistatin, laminin, and perlecan
• Isolated due to its ability to cluster pre-existing
acetylcholine receptors in skeletal muscle fibers
• BM form binds to laminin
Agrin
• Several splice variants exist; critical for function in skeletal muscle.
– The “Z” exon is present in nerve-derived but not musclederived agrin and is necessary for its AChR clustering activity.
• Agrin may be the most dramatic example of a basement
membrane component with a specific, well-defined signaling role.
The Collagens
•
•
•
•
The most ubiquitous structural protein.
Characterized as a triple helical protein
containing peptide chains with repeating
Gly-Xaa-Yaa (usually Pro) triplets.
The triple helix forms through the
association of three related polypeptides
( -chains) forming a coiled coil, with the
side chain of every third residue directed
towards the center of the superhelix.
Steric constraints dictate that the center of
the helix be occupied only by Glycine
residues.
Many Proline and Lysine residues are
enzymatically converted to hydroxyproline
and hydroxylysine.
~28 distinct collagen types; each is
assigned a Roman numeral that generally
delineates the chronological order in
which the collagens were
isolated/characterized.
Diversity of Collagens
Type I
fibrils
Skin, tendon, bone, ligaments, dentin,
interstitium
Type II
Fibrils
Cartilage, vitreous humor
Type III
Fibrils
Skin, muscle, bv
Type IV
2D sheets
All basement membranes
Type V
Fibrils with
globular end
Cornea, teeth, bone, placenta, skin,
smooth muscle
Type VI
Fibril-assoc. (I)
Most interstitial tissues
Type VII
Long anchoring
fibril
Skin--connects epidermal basement
membrane/hemidesmosome to dermis
Type IX
Fibril-assoc. (II)
Cartilage, vitreous humor
Type XIII
Transmembrane
Hemidesmosomes in skin
Type XV
HSPG
Widespread; near basement
membranes in muscle
Type XVII
Transmembrane
Hemidesmosomes in skin (aka BPAG2
or BP180)
Collagen IV: Network or Sheet Forming
• Six genetically distinct  chains: α1- α6, ~180 kDa each
• Chains form three types of heterotrimers:
– ( 1)2(α2), α3α4α5, (α5)2(α6)
• Like all Collagens, comprised mainly of Gly-x-y repeats, y is
frequently proline
• Gly-x-y pattern has multiple interruptions
– Provides flexibility to the collagen network and to the basement
membrane
Hudson et al., NEJM 2003
Collagen IV Trimer
• 7S domain at N-terminus
• Interrupted Gly-x-y triple
helical domain
• C-terminal non-collagenous
domain--NC1
Collagen IV Network
Trimers (aka protomers)
associate with each other,
four at the N-terminus and
two at the C-terminus
(hexamer), to form a
chicken wire-like network
that provides strength and
flexibility to the basement
membrane.
What Directs Chain-Chain-Chain
Recognition and Hexamer Assembly?
Sulfilimine: The Bond that Crosslinks
Type IV Collagen NC1 Domains
Vanacore et al., Science 2009
Type IV Collagen Mutations and
Human Disease
• COL4A1 mutations
– Small vessel disease/retinal vascular
tortuosity
– Hemorrhagic stroke
– Porencephaly
Kidney Glomerular BM
– HANAC syndrome
• COL4A3/A4/A5 mutations
– Alport syndrome/hereditary
glomerulonephritis
Fibrillar Collagens (I, II, III, V)
Fibrillar Collagens (I, II, III, V)
• Connective tissue proteins that
provide tensile strength
• Triple helix, composed of three
 chains
• Glycine at every third position
(Gly-X-Y)
• High proline content
– Hydroxylation required for proper
folding and secretion
• Found in bone, skin, tendons,
cartilage, arteries
Biosynthesis of Fibril-forming Collagens
Prolyl hydroxylases
Lysyl hydroxylase
Glycosyltransferases
Procollagen N- and Cproteinases
Lysyl oxidase
Adapted from: Keilty, Hopkinson, Grant. In: Connective Tissue and
Its Inheritable Disorders, Wiley-Liss, 1993.
Collagen Crosslinking
•
•
•
Once formed, collagen fibrils are greatly strengthened by covalent crosslinks that
form between the constituent collagen molecules.
The first step in crosslink formation is the deamination by the enzyme lysyl
oxidase of specific lysine and hydroxylysine side chains to form reactive aldehyde
groups.
The aldehydes then form covalent bonds with each other or with other lysine or
hydroxylysine residues.
Collagen Crosslinking
•
•
If crosslinking is inhibited (Lysyl
hydroxylase mutations; vitamin C
deficiency), collagenous tissues
become fragile, and structures
such as skin, tendons, and blood
vessels tend to tear. There are
also many bone manifestations of
under-crosslinked collagen.
Hydroxylation of specific lysines
governs the nature of the crosslink formed, which affects the
biomechanical properties of the
tissue. Collagen is especially
highly crosslinked in the Achilles
tendon, where tensile strength is
crucial.
Bone is Composed of Mineralized
Type I Collagen Fibrils
Bone is 70%
mineral and 30%
protein, mostly
collagen
Mineral is Dahllite,
similar to
hydroxyapatite
(contains calcium,
phosphate,
carbonate)
Scurvy
• Liver spots on skin, spongy
gums, bleeding from mucous
membranes, depression,
immobility
• Vitamin C deficiency
• Ascorbate is required for prolyl
hydroxylase and lysyl
hydroxylase activities
• Acquired disease of fibrillar
collagen
Illustration from Man-of-War by Stephen Biesty (Dorling-Kindersley, NY, 1993)
Some Genetic Diseases of Collagen
• Collagen I
– Osteogenesis imperfecta
– Ehlers-Danlos syndrome type VII
• Collagen II
– Multiple diseases of cartilage
• Collagen III
– Ehlers-Danlos syndrome type IV
• Collagen IV
– Alport syndrome, stroke, hemorrhage, porencephaly
• Collagen VII
– Dystrophic epidermolysis bullosa (skin blistering)
Different Types of Mutations in Collagen I  Chain
Genes Cause Different Disease Severities
Gene
location mutation
Syndrome
COL1A1
17q22
Null alleles
OI type I
Partial deletions; C-terminal
substitutions
OI type II
N-terminal substitutions
OI types I, III or IV
Deletion of exon 6
EDS type VII
COL1A2
7q22.1
Splice mutations; exon deletions OI type I
C-terminal mutations
OI type II, IV
N-terminal substitutions
OI type III
Deletion of exon 6
EDS type VII
Osteogenesis Imperfecta
(brittle bone disease)
Clinical:
Ranges in severity from mild to perinatal lethal
bone fragility, short stature, bone deformities, teeth
abnormalities, gray-blue sclerae, hearing loss
Biochemical:
reduced and/or abnormal type I collagen
Molecular:
mutations in either type I collagen gene, COL1A1 or COL1A2,
resulting in haploinsufficiency or disruption of the triple helical
domain (dominant negative: glycine substitutions most common)
COL1 Haploinsufficiency (Dominant)
(α1)2α2
Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.
Dominant Negative COL1 Mutations
½ of the
trimers are
abnormal
*
Gly subst. in COL4A2
*
Gly subst. in COL4A1
¾ of the
trimers are
abnormal
Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50.
Elastin and Elastic Fibers Exhibit
Rubber-Like Properties
•
•
•
Physiological importance lies in the unique
elastomeric properties of elastin. Found in
tissues in which reversible extensibility or
deformability are crucial, such as the major
arterial vessels (esp. aorta), the lung and the
skin.
Elastin is characterized by a high index of
hydrophobicity (90% of all the amino acid
residues are nonpolar). One-third of the
amino acid residues are glycine with a
preponderance of the nonpolar amino acids
Ala, Val, Leu, and Ile. As in collagen, oneninth of the residues are proline (but with very
little hydroxylation).
Early in development, the elastic fibers
consists of microfibrils, which define fiber
location and morphology. Over time,
tropoelastin accumulates within the bed of
microfibrils.
Elastic Fiber Biogenesis
•
•
Elastic fibers are very complex,
difficult to repair structures
There are two morphologically
distinguishable components
–
–
•
Microfibrils
Elastin
Assembly follows a well-defined
sequence of events:
1. Assembly of microfibrils
2. Association of tropoelastin aggregates with
microfibrils
3. Crosslinking of tropoelastins with each
other by lysyl oxidase to form polymers
Shifren and Mecham, 2006
Major steps underlying the assembly
of microfibrils and elastic fibers
Ramirez, F. et al. Physiol. Genomics 19: 151-154 2004;
doi:10.1152/physiolgenomics.00092.2004
Copyright ©2004 American Physiological Society
Microfibril Components: ~30
• Fibrillin--three forms
• Microfibril-associated glycoproteins
(MAGPs)--two forms
• Latent TGFBinding Proteins
(LTBPs)--four forms
• Proteoglycans, MFAPs, Fibulins,
Emilins, Collagens, Decorin, et al.
Fibrillin-1
RGD
Fibrillin -2
Gly
RGD
Fibrillins
Fibrillin -3
P/G
LTBP-1
Fibrillin-1
RGD
RGD
EGF
P ro
Fibrillin -2
RGD
LTBP-2
Gly
Hybrid (CC)
Unique
Glycosylation (potential)
RGD
LTBP-3
Fibrillin -3
EGF--Ca Binding
8-Cys (CCC)
RGD
P/G
RGD
LTBP-4
LTBP-1
RGD
EGF
EGF--Ca Binding
• Large
glycoproteins
(~350
kDa)
whose
primary
8-Cys (CCC) structures are
LTBP-2
2+,
(CC)
dominated by cbEGF domains that, in theHybrid
presence
of
Ca
Unique
LTBP-3 a rodlike structure
Glycosylation (potential)
adopt
• Limited
intracellular assembly may occur, but microfibril
LTBP-4
assembly initiates at the cell surface after secretion, perhaps
with the help of cellular receptors
RGD
RGD
Marfan Syndrome
• Caused by dominant Fibrillin1 (FBN1) mutations
– Haploinsufficiency is the culprit
• Skeletal, ocular, and
cardiovascular defects
• Deficiency of elastinassociated microfibrils
• Syndrome may result from
alterations in TGFsignaling,
rather than purely structural
changes in microfibrils
Latent TGFBinding Proteins
• Members of the fibrillin superfamily
• Maintain TGFin the inactive state by
forming the “large latent complex”
RGD
Fibrillin-1
P ro
RGD
Fibrillin -2
Gly
RGD
Fibrillin -3
P/G
RGD
LTBP-1
EGF
RGD
LTBP-2
EGF--Ca Binding
8-Cys (CCC)
Hybrid (CC)
Unique
Glycosylation (potential)
LTBP-3
RGD
LTBP-4
The TGFLarge Latent Complex (LLC)
Potential Activators:
ROS
Proteases
Integrins
(Assoc. with ECM
Perturbations)
(e.g., fibrillin)
LAP: Latency-associated
peptide
Annes, J. P. et al. J Cell Sci 2003;116:217-224
Evidence for FBN/BMP7 Interactions
Fbn2+/-; Bmp7+/- transheterozygous animals
show limb patterning
defects.
Artaga-Solis et al., J. Cell Biol. 2001
Specific
fragments of
Fibrillin 1, but
not LTBP1,
bind to BMP7
Gregory et al., JBC 2005
Elastic Fiber Biogenesis
•
•
Elastic fibers are very complex,
difficult to repair structures
There are two morphologically
distinguishable components
–
–
•
Microfibrils
Elastin
Assembly follows a well-defined
sequence of events:
1. Assembly of microfibrils
2. Association of tropoelastin aggregates with
microfibrils
3. Crosslinking of tropoelastins with each
other by lysyl oxidase to form polymers
Shifren and Mecham, 2006
Emphysema
• Damage to the lung air sacs
(alveoli) that affects breathing
• Macrophages induced to “ingest”
particles in smoke also secrete
proteases that degrade elastic
fibers
• Loss of lung elasticity makes
exhalation difficult
• Increased alveolar size reduces
the surface area for gas exchange