Lauren Canova Lucy Greetham Mark Nowey Casey Pham Britney Tappen 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 What processes do you think these interactions are necessary for?

What is the importance of the ECM? http://ib-bio.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, 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

http://www. rcsb. org/pdb/explore/jmol. do http://www.rcsb.org/pdb/explore/jmol.do?structureId=3ADM&bionumber=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 C-proteinase Self-assembly due to hydrophobic, electrostatic and cross-linking 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

Non-helical telopeptide Gap region Overlap region

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

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. http://skillbuilders.patientsites.com/article.php?aid=253 http://www.theherbprof.com/ailArthritis.htm

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?)

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…

Ehlers–Danlos syndrome – Type with Hypermobility 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

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)