1. Lauren Canova Lucy Greetham Mark Nowey Casey Pham Britney Tappen
Collagen
Lauren Canova
Lucy Greetham
Mark Nowey
Casey Pham
Britney Tappen

2. 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)

3. Lets see what you know…
List all the cell- ECM interactions that you can think of
What processes do you think these interactions are necessary for?

4. What is the importance of the ECM?

6. 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,
Provides structural stability because of crosslinking of hydroxyl groups between microfibrils
Triple helix provides strength and resistance against most proteases including pepsin, trypsin or
chymotrypsin

7. http://www. rcsb. org/pdb/explore/jmol. do

8. 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

9. 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

10. Collagen Biosynthesis
Collagen molecules made of 3 α-chains
Modified in Golgi
Cleavage of propeptides by N-proteinase and C-proteinase
Self-assembly due to hydrophobic, electrostatic and cross-linking interactions
5 collagen molecules form a fibril, which associate into fibers

12. Secondary Structure Adopts a Triple Helix Formation.
3 LEFT HANDED Helices form a
RIGHT HANDED
SUPER HELIX.
Voet, Fundamentals of Biochemistry, 3/e

13. Non-helical telopeptide
Gap region
Overlap region

14. Hierarchal structure
Helical structure enclosed with non-helical telopeptides
Telopeptides are the sites of intermolecular cross-linking
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

16. Collagen Crosslinking
Power in Numbers! Fiber = Strength Fibril = Weak
Collagen Crosslinking
Much of the tensile strength of collagen is due to CROSSLINKED fibrils into strong FIBERS.

17. Collagen Structure Imino Modifications Primary Structure:
Gly – X – Y Motif
Imino Modifications
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?)

18. 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

19. End up with this after crosslinking…

20. Ehlers–Danlos syndrome – Type with Hypermobility

21. 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.

23. 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

24. 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

25. 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

26. 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

27. Cleavage site on monomer 4
E. Yellow arrow= removal of C terminal telopeptide from monomer 5, which allows access to MMP1

28. 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

29. 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

30. 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?

31. 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

33. 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

34. 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

35. Activity 3: Part 2
Arrange the collagen molecules to represent the structure of a collagen fibril with D periodicity
Show crosslinking between monomers

36. Activity 3: Part 3
Remove the part of the fibril which must be cleaved to allow MMP1 access to its cleavage site.

37. 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)