Transcript Collagen

Lauren Canova
Lucy Greetham
Mark Nowey
Casey Pham
Britney Tappen
Learning Objectives
 Identify the primary constituents of collagen fibrils
and recognize hierarchal organization (Bloom’s
Cognition Level 1, Remember)
 Predict the phenotypic outcome on a cell, tissue, and
organism caused by a change in the structure of
collagen (Bloom’s Cognition Level 2, Understand)
 Hypothesize the mechanism of a collagen related
disorder (Bloom’s Cognition Level 6, Create)
 Create a model of collagen (Bloom’s Cognition Level 6,
Create)
Lets see what you know…
List all the cell- ECM interactions that you can think
of
2. What processes do you think these interactions are
necessary for?
1.

What is the importance of the ECM?
http://ibbio.wikispaces.com/Biology+Drawings
http://www.biolsci.org/v03p0303.htm
Micro structure
Basic structure
Three left helices for right handed triple helix
Gly-X-Y sequence,
Why?
Smallest amino acid allows for tight packing
Another common amino acids include hydroxyproline,
Why?
Provides structural stability because of crosslinking of
hydroxyl groups between microfibrils
http://www.rcsb.org/pdb/explore/jmol.do?structureId=3ADM&bion
umber=1
Collagen
Strength – Triple helix provides tensile strength
Scaffold – Provides organization and structure for the ECM
Without it, what would happen?



Loss of cell-cell communication
Cell migration
Loss of cell shape
Collagen Biosynthesis
 Fibroblasts and other cells secrete
collagen creating their own ECM
and microenvironment
 Factors that Promote Synthesis of
Collagen
 TGF-β
 Insulin-like growth factors
 Fibroblast growth factors
 Collagen can bind cytokines and
other growth factors
 Cells can regulate the structure of
collagen and therefore the
availability of these factors
 Example of feed back loop
http://course1.winona.edu/sberg/IMAGES/fibroblast.jpg
Collagen Biosynthesis Collagen molecules




made of 3 α-chains
Modified in Golgi
Cleavage of propeptides
by N-proteinase and Cproteinase
Self-assembly due to
hydrophobic,
electrostatic and crosslinking interactions
5 collagen molecules
form a fibril, which
associate into fibers
Secondary Structure
Adopts a Triple Helix
Formation.
3 LEFT HANDED
Helices form a
RIGHT HANDED
SUPER HELIX.
Voet, Fundamentals of Biochemistry, 3/e
http://www.biomed.curtin.edu.au/biochem
/tutorials/prottute/helixfigures.htm
Overlap
region
Gap
region
Nonhelical
telopeptid
e
Hierarchal structure
 Helical structure enclosed with non-helical
telopeptides
 Telopeptides are the sites of intermolecular crosslinking
 5 “rows” of molecules with staggered stacking leads to:
 Gap region where there are 4 molecules (leaves space for
stain, dark region)
 Overlap region where there are 5 molecules
 Together make the D-period
 Collagen fibrils combine to form collagen fibers
Collagen
Crosslinking
Much of the tensile
strength of collagen is
due to CROSSLINKED
fibrils into strong
FIBERS.
Power in Numbers!
Fiber = Strength
Fibril = Weak
http://www.theherbprof.com/ailArthritis.h
tm
http://skillbuilders.patientsites.com/a
rticle.php?aid=253
Collagen Structure
Primary Structure:
Gly – X – Y Motif
 Requires Glycine every third
residue.
 X is often Proline
 Y is often Hydroxyproline
 Hydroxyproline and
Hydroxylysine formed from
modification of Proline
AFTER synthesis
 Hydroxyproline formation
requires Vitamin C cofactor
(scurvy anyone?)
Imino Modifications
Crosslinking
Mechanism
HYDROXYLATED LYSINE RESIDUES
FOUND IN TELOPEPTIDE SECTION
UNDERGO ENZYMATIC TREAMENT AND
CROSSLINK TO ADAJACENT FIBRILS.
Key Enzyme:
Lysyl Oxidase
Adol- His can then react with Hyl imino
group of neighboring fibril to complete the
crosslinkage
Voet, Fundamentals of
Biochemistry, 3/e
End up with this after crosslinking…
http://www.whatsonxiamen.com/news10636.html
Activity 1 (10 Minutes)
 Your friend Fred loves eating Chickling peas, which he
gets mail-ordered from Asia.
 Lately, Fred has been feeling ill, with pain in his joints
and numbness in his lower extremities
 The Doctor told Fred to take some vitamin-C, but that
didn’t help.
 Fred asks for your opinion, since you have studied
collagen. Split up into groups of 4 or 5 and write down
some possible causes of Fred’s ailments.
Activity 1 (Answer!)
 Fred suffers from Osteolathyrism
 This disorder is caused by a toxin in the chickling pea
which inactivates lysyl oxidase
 Due to this inactivation, collagen cannot cross-link,
and causes weak bones, pain in joints, and numbness
Matrix Metalloproteinases (MMP)
 Responsible for Catabolism of ECM
 Connective tissue remodeling
 Development
 Wound healing
 Tumor cell invasion and metastasis
 At least 25 types, categorized by domain structure and
macromolecular preference
 Limiting factor is the ability to properly orient and
potentially destabilize collagen
 Either collagen must unwind on its own, backbone
mobility, or the protease unwinds the collagen
 unknown
 MMP1 and 8 have catalytic domain exosite, substrate
binding site far from active site, which acts as triple
helix recognition
 MMP3 lacks this domain, and can cleave only single
molecules but not native triple helix
Cleavage Site ~25 amino acid
sequence
 MMP 1 preferentially binds monomer 4
 Other monomers have same sequence (may facilitate
recognition)
 Cleavage site Gly-Pro-Gly775~Ile 776- Ala-Gly-Gln
A. Cleavage site on monomer 4
E. Yellow arrow= removal of C terminal telopeptide from monomer 5, which
allows access to MMP1
http://www.pnas.org/content/105/8/2824.full
 Integrins are heterodimeric cell surface receptors, α1β1
binds collagen 1
 Connect cytoskeleton to ECM
 Regulate cell shape, orientation, and migration
 Influence cell-cell signaling and differentiation
 Inside-out or outside-in
 Poorly understood
Changes in the
structure of the ECM
will propagate signals
to the interior of the
cell
B. single D period of microfibril = 5 triple
helix
C terminal telopeptide forms bulging ridge
C. Red arrow shows region where C
terminal telopeptide has been removes to
allow access to MMP1 to cleavage site
(yellow band), the rest of monomer 4 is
dark grey
Integrin Binding site shown in green
demonstrates why there is poor integrin
binding to intact fibrils because both α1
chains are inaccessible. M5 and M4 need to
be removed
E. Red arrow shows MMP cleavage site
Green arrow shows integrin binding site
http://www.pnas.org/content/105/8/2824.full
Activity 2 (10 min)
 1. How would a mutation to the exocite of MMP1 ,
which recognizes the native triple helical structure of
collagen, effect the cell, tissue and organism?
 2. There was a mutation in the promoter of the MMP1
gene, causing its overexpression. How would this effect
the cell, tissue, and organism?
Answer
 1. This would mean that MMP1 would be unable to
cleave native collagen causing problems in
development, wound healing, angiogenesis, etc.
 Potentially fatal in early development
 2. This would cause the cell to excessively degrade its
microenvironment. Depending on the degree, this
could expose normally hidden binding sites, allow for
increase locamotion, or could lead to tissue
degradation
Overall View of Collagen in the
ECM
 The cell synthesized and secretes collagen, which
creates a microenvironment for the cell to inhabit.
 This microenvironment regulates and influences cell
function
 Feedback Loop = ECM- Cell
Activity 3: Part 1
 Get into groups of 5
 Twizzlers strands (cherry) represent α chains
 Straws are to be used as an arbitrary backbone
 Construct a collagen molecule and label the N and C
terminus
Activity 3: Part 2
 Arrange the collagen molecules to represent the
structure of a collagen fibril with D periodicity
 Show crosslinking between monomers
Activity 3: Part 3
 Remove the part of the fibril which must be cleaved to
allow MMP1 access to its cleavage site.
Learning Objectives
 Identify the primary constituents of collagen fibrils
and recognize hierarchal organization (Bloom’s
Cognition Level 1, Remember)
 Predict the phenotypic outcome on a cell, tissue, and
organism caused by a change in the structure of
collagen (Bloom’s Cognition Level 2, Understand)
 Hypothesize the mechanism of a collagen related
disorder (Bloom’s Cognition Level 6, Create)
 Create a model of collagen (Bloom’s Cognition Level 6,
Create)