CSB327 Lecture 3 Notes

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Department
Cell and Systems Biology
Course
CSB327H1
Professor
Maurice Ringuette
Semester
Fall

Description
CSB327 Lecture 3 Notes – Extracellular processing (September 17, 2012) 2 – Lecture outline • The next step is to look at extracellular fibrillogenesis that happens on the outside of the cell. You cleave the N- and C- pro-peptide within the matrix compartment but it is on the extracellular side of the cellular compartment. 3 – Cross-links formed between modified lysine side chains with a collagen fibril • You have two types of cross linking. You have intramolecular cross linking which is within the triple helix. You have intermolecular cross linking which is between C- terminal and N-terminal telopeptides. You get more cross linking between telopeptides. What is the enzyme responsible for this? 4 – Side-by-side interactions between collagen molecules is stabilized by aldol cross-links between two lysine side chains • I will not ask you to re-draw Lys residue. You can have cross linking in the telopeptide domain and to a lesser degree, within the triple helix of Lys and HO-Lys residues. The enzyme that does this is lysyl oxidase (LO). I want you to know that you have a delamination reaction and you create highly reactive aldehyde intermediates that can spontaneously form an aldol cross link. I want you to appreciate lysyl oxidase, requiring oxygen, is responsible for the cross linking of collagen molecules together. The pro form of the enzyme (Pro-LO) is cleaved into LO. The pro domain has biological activity. One of the problems with cancer is that you have a process called fibrosis, which is an excessive deposition of ECM molecules like Type I collagen and an excessive production of LO that cross links them. LO helps increase the strength of a tumour by excessive cross linking. LO has huge applications for other things. What do I expect you to remember? The enzyme that is responsible, where it is and what its function is. • Remember that glycosylation of Type I collagen could do with the turnover of the matrix after it is degraded by metalloproteinases. If you have the right level of glycosylation, then it will prevent excessive cross linking of collagen. • HO-Lys cross linking forms more stable bonds than Lys cross linking. 5 – Lysine aldehyde pathway • You see Lys cross linking mostly in the skin, cornea and sclera. You HO-Lys cross linking in connective tissues such as bone, cartilage, etc. This is for your information only at the moment. I just want to point out that there are different isoforms of LO. There are up to five different LO in our bodies. 6 – The structure of fibrillar collagens: molecule  fibrils  fibers • You have a right handed triple helix staggered by quarter length. Between the C- terminus of this collagen molecule and that collagen molecule is about 67-70 nm. This is repeated because they are staggered by this amount. They are cross linked so they will have incredible tensile strength. There is a small 40 nm gap between them which becomes important. Here is a TEM to show you that you see the repeated pattern because it is due to the gap overlap region. One D-period is 67 nm. A collagen molecule is about 4.4 D. 7 – Schematic representation of the supramolecular assembly of the collagen fibrils in the characteristic quarter-staggered form • The region between the gap of one molecule and the overlap has about six collagen molecules that are solid. You can see that some gaps occur within the six collagens because of the way that they are staggered. This has issues that will come up that I want to emphasize. 8 – Electron micrographs of native collagen fibrils stained with either positive or negative stain • Let us focus on the negative stain. The important thing is that because of the staggering, wherever you have a hole zone, then you take up more dye. Under the TEM, then you will see a black band. Many molecules stagger upon each other. In the overlap zone, you take up less dye. You then bump into another hole zone and another overlap zone and so on. This is why you see an alternating stains between hole zone and overlap zone when you use negative stain in TEM. The hole zone is about 0.6 D. The overlap zone is about 0.4 D. In positive stain, you target the polar amino acids. You can see a repeated pattern because it is the same type of molecule staggered along the fibril. It is almost like a barcode. If you use a different type of collagen with a different organization of the polar amino acids, then you would see a difference. Often when you overlap them, you see the repeated pattern as seen with the positive stain and negative stain. • We talked about the periodic nature of the staggering which has a gap. Then you had the next molecule and another gap. When you staggered them, there were regions where there was complete overlap and there were regions of every six where there was a gap. I talked about staining it. I talked about this 1D overlap period of 67 nm. 9 – Collagen fibers • You can see the repeated pattern under simple light microscopy or TEM. • The half life of most collagen fibrils is about 6 months. But in tissues, this is a deceiving number because when do you get scurvy? In about 2 months. A lot of collagens turnover faster than the half life. Skin and vasculature remodelling is fast. Bone and ligament remodelling is much slower. Tissues that are more dynamic in nature in terms of remodelling can turnover quickly. Turnover is accomplished by matrix metalloproteinases. 10 – Function of hole zones? • What is the purpose of the hole zones? One is access to modifying enzymes. As we saw, as we go through collagen fibrillogenesis in extracellular compartments, you have access to LO. The second thing is that they tend to be the site of mineralization first in osteogenesis. The third is fibril flexibility. Think of the bones being made of calcium phosphate. You have a rod shaped molecule that has a lot of tensile strength when cross linked. The molecule must be able to bend a certain degree so that it doesn’t snap. The hole zone is also important to give a little bit of crimping in the ligaments so that you can pull to a certain degree before you snap the ligaments. The crimping because of the hole zone helps give fibril flexibility. 11 – Ehlers-Danlos Syndrome (EDS) • EDS is associated with failure to process Type I collagen. EDS is correlated with some event of collagen either mutation of collagen or processing of collagen. The matrix is involved in regulating all these facets. We are going to build onto the collagens with other molecules and I will use a prototypic disease to emphasize how these two act in concert. 12 – EDS classifications • I want to talk to you about another pathology that falls under the umbrella of EDS. Dematosparaxis disease has to do with a mutation or deficiency in ADAMTS2. What kind of molecule is ADAMTS2? ADAMTS2 is a matrix metalloprotease, so it is going to target matrix molecules. I am going to go into more detail about matrix remodelling metalloproteases. I will give you more details about this and what is special about these enzymes. We will look at a classical type of EDS. You do not have to worry about the rest of them but you can
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