2Learning ObjectivesIdentify 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)
3Lets see what you know…List all the cell- ECM interactions that you can think ofWhat processes do you think these interactions are necessary for?
6Micro structure Basic structure Three left helices for right handed triple helixGly-X-Y sequence,Why?Smallest amino acid allows for tight packingAnother common amino acids include hydroxyproline,Provides structural stability because of crosslinking of hydroxyl groups between microfibrilsTriple helix provides strength and resistance against most proteases including pepsin, trypsin orchymotrypsin
8Collagen Strength – Triple helix provides tensile strength Scaffold – Provides organization and structure for the ECMWithout it, what would happen?Loss of cell-cell communicationCell migrationLoss of cell shape
9Collagen Biosynthesis Fibroblasts and other cells secrete collagen creating their own ECM and microenvironmentFactors that Promote Synthesis of CollagenTGF-βInsulin-like growth factorsFibroblast growth factorsCollagen can bind cytokines and other growth factorsCells can regulate the structure of collagen and therefore the availability of these factorsExample of feed back loop
10Collagen Biosynthesis Collagen molecules made of 3 α-chainsModified in GolgiCleavage of propeptides by N-proteinase and C-proteinaseSelf-assembly due to hydrophobic, electrostatic and cross-linking interactions5 collagen molecules form a fibril, which associate into fibers
12Secondary Structure Adopts a Triple Helix Formation. 3 LEFT HANDED Helices form aRIGHT HANDEDSUPER HELIX.Voet, Fundamentals of Biochemistry, 3/e
13Non-helical telopeptide Gap regionOverlap region
14Hierarchal structureHelical structure enclosed with non-helical telopeptidesTelopeptides are the sites of intermolecular cross-linking5 “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 moleculesTogether make the D-periodCollagen fibrils combine to form collagen fibers
16Collagen Crosslinking Power in Numbers! Fiber = Strength Fibril = WeakCollagen CrosslinkingMuch of the tensile strength of collagen is due to CROSSLINKED fibrils into strong FIBERS.
17Collagen Structure Imino Modifications Primary Structure: Gly – X – Y MotifImino ModificationsRequires Glycine every third residue.X is often ProlineY is often HydroxyprolineHydroxyproline and Hydroxylysine formed from modification of Proline AFTER synthesisHydroxyproline formation requires Vitamin C cofactor (scurvy anyone?)
18Crosslinking Mechanism HYDROXYLATED LYSINE RESIDUES FOUND IN TELOPEPTIDE SECTION UNDERGO ENZYMATIC TREAMENT AND CROSSLINK TO ADAJACENT FIBRILS.Key Enzyme:Lysyl OxidaseAdol- His can then react with Hyl imino group of neighboring fibril to complete the crosslinkageVoet, Fundamentals of Biochemistry, 3/e
20Ehlers–Danlos syndrome – Type with Hypermobility
21Activity 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 extremitiesThe 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.
23Activity 1 (Answer!) Fred suffers from Osteolathyrism This disorder is caused by a toxin in the chickling pea which inactivates lysyl oxidaseDue to this inactivation, collagen cannot cross-link, and causes weak bones, pain in joints, and numbness
24Matrix Metalloproteinases (MMP) Responsible for Catabolism of ECMConnective tissue remodelingDevelopmentWound healingTumor cell invasion and metastasisAt least 25 types, categorized by domain structure and macromolecular preference
25Limiting 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 collagenunknownMMP1 and 8 have catalytic domain exosite, substrate binding site far from active site, which acts as triple helix recognitionMMP3 lacks this domain, and can cleave only single molecules but not native triple helix
26Cleavage Site ~25 amino acid sequence MMP 1 preferentially binds monomer 4Other monomers have same sequence (may facilitate recognition)Cleavage site Gly-Pro-Gly775~Ile 776- Ala-Gly-Gln
27Cleavage site on monomer 4 E. Yellow arrow= removal of C terminal telopeptide from monomer 5, which allows access to MMP1
28Integrins are heterodimeric cell surface receptors, α1β1 binds collagen 1 Connect cytoskeleton to ECMRegulate cell shape, orientation, and migrationInfluence cell-cell signaling and differentiationInside-out or outside-inPoorly understoodChanges in the structure of the ECM will propagate signals to the interior of the cell
29B. single D period of microfibril = 5 triple helix C terminal telopeptide forms bulging ridgeC. 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 greyIntegrin 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 removedE. Red arrow shows MMP cleavage siteGreen arrow shows integrin binding site
30Activity 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?
31Answer1. This would mean that MMP1 would be unable to cleave native collagen causing problems in development, wound healing, angiogenesis, etc.Potentially fatal in early development2. 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
33Overall 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 functionFeedback Loop = ECM- Cell
34Activity 3: Part 1 Get into groups of 5 Twizzlers strands (cherry) represent α chainsStraws are to be used as an arbitrary backboneConstruct a collagen molecule and label the N and C terminus
35Activity 3: Part 2Arrange the collagen molecules to represent the structure of a collagen fibril with D periodicityShow crosslinking between monomers
36Activity 3: Part 3Remove the part of the fibril which must be cleaved to allow MMP1 access to its cleavage site.
37Learning ObjectivesIdentify 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)