CSB327 Lecture 2 Notes

9 Pages
Unlock Document

University of Toronto St. George
Cell and Systems Biology
Maurice Ringuette

CSB327 Lecture 2 Notes – Molecular organization and classification of collagens (September 13, 2012) 1 – Lecture topics  Type I collagen will be used as the prototype for fibrillar collagens.  Type I collagen is the dominant collagen in the skin and bone. 3 – Supramolecular Assemblies of Collagens based on rotary shadowing electron microscopy  This is an overview. Different collagens are classified by the superstructures that they form. You can see structural diversity between collagens. Type IV collagen is the signature collagen of basement membrane, which forms a network. FACIT collagens interact with Type I collagen and promote proper assembly of fibrils. 4 – Classification of Collagens  I want to emphasize the nomenclature. The chain composition of Type I collagen is 1,1,2. This means that Type I collagen is a blend of α1 and α2 chain. This means that you have a heterogeneous polymer. The molecular weight shows you that they are the same length in the fibril analysis. Type I is almost ubiquitous, but more abundant in bone and so on. α1(I)2α2(I) heterotrimer means you have two α1-chains of Type I collagen and one α2-chain of Type I collagen. There are two transcriptional units that are responsible for the formation of Type I collagen molecule. The type of collagen is in brackets.  Type II collagen is the signature collagen of cartilage. It is a homotrimer, which means there is only one gene that codes for Type II collagen. There are three α1-chains of Type II collagen.  Type III collagen is α1(II3) homotrimer. 5 – Fibrillar collagens  Type V collagen is a nucleator. 6 – Genetic Organization of Type I Collagen  You do not need to memorize this. There is N-propeptide and C-propeptide for Type I collagen. Exon duplication created tandems of Gly-X-Y. Type I collagen is made of two α1-chains and one α2-chains. The N-terminal globular domain and the C-terminal domain must be cleaved in the extracellular space for collagen fibrils to form. If you cleave them prematurely, then it can self-polymerize inside the cell. It is fundamental that the molecule does not pre-assemble into a polymer inside the cell, so there are globular structures at the ends. By this stage, the signal peptide is not attached anymore. You are shooting a collagen molecule with intact C- and N- termini into the ER. Telopeptides are important. 7 – Fibroblast cell imaged in culture medium  The major cell that make connective tissue are fibroblast cells. Fibroblast cells are non- epithelial cells that do not have the polarized character of epithelial cells. They are a major source of ECM molecule in our hearts. The cardio fibroblast cells make collagen and ECM functional. Fibroblast cells refer to a cell type that makes a major amount of ECM. It can be adhesive and they are morphologically heterogeneous. 8 – Overview of fibrillar collagen synthesis and assembly  α-chain  hydroxylation  triple helix  make its way out of secretory pathway through Golgi into extracellular space  collagen molecule gets cleaved  form staggered and repeated arrangement  fibrils  fibres  Pathologies of collagen molecules refer to collagen molecules after secretion. Defects here have lead to consequences of poor bone and poor skin formation. A mutation here has dramatic effects at this end. It is an epistatic event. 9 – Collagen Biosynthesis Pathway  You would see vesicles like the ER, Golgi network, and collagen fibrils. Unless otherwise mentioned, I am going to talk about collagen fibrils most of the time. You can see distinct collagen fibrils. The orientation is in a weave-like fashion. The collagen fibrils are uniformly sized. The fibrils are not all contacting each other. The fibrils are not fused. 10 – Tissue specific three dimensional (3D) arrays of type I collagen  You tend to see the fibrils, assembled into fibers, in a specific orientation. The cornea has to be transparent and precisely organized. One of the major biochemical properties of collagen is tensile strength. Collagen fibre is tougher than steel. Bones are formed in concentric rings of ECM of mineralization. Our skin is weave-like. The three-dimensional organization reflects the direction of the force. Approximately 3 kg of our body weight is collagens. 11 – Post-translational modifications of collagens in the endoplasmic reticulum  Formation of protein aggregates (monomeric complexes) happens in the ER. 12 – Isomerization of proline residues in the ER: a rate limiting step  The Pro residue is in the cis conformation and needs to be changed into the trans conformation for it to form the polyproline left-handed helix as an α-chain that will be transformed into a right-handed triple helical structure. Without this step, you have a right-handed helix that doesn’t properly assemble into a collagen molecule. Pro residues can bend the chain. This is a rate limiting step, as synthesis of α-chains occurs in the ER, PPI will convert it into an isoform that is capable of assembling into a helix. Without PPI, you cannot make collagen. What is the biological significance of PPI? It is a rate limiting step. 13 – ER Processing of Fibrillar Collagens  The next major event that occurs is the formation of hydroxyl groups that are added to the chain as synthesis occurs. Note that the signal peptides are already cleaved off. As you make an α-chain, it becomes hydroxyl. X can be Pro but it is rare. You cannot assemble a collagen molecule from Pro-α-chains. You cannot assemble a collagen molecule until the C-terminal region has been made. If you miss hydroxylation, the helix is unstable at our body temperature. Hydroxylation is to stabilize the triple helix. 14 – Hydroxylation of proline and lysine residues  Hydroxylation is critical. Prolyl-3-hydroxylase and Prolyl-4-hydroxylase is responsible for the hydroxylation of Pro residues. Prolyl-4-hydroxylase is the predominant one. As primates, we cannot make vitamin C, but it is co-factor that is required by these enzymes. I will focus on the vitamin C aspect. 15 – Hydroxylation of Collagen Proline Residues in the ER  Pro-986 is conserved in evolution and defects in hydroxylation cause mutations also. 4- hyroxyproline is the predominant form. Hydrogen bonds from Gly and HO-Pro residues are responsible for the stability of the helix. 17 – Scurvy  Scurvy is due to a lack of vitamin C. This was a big problem for sailors. You get vitamin C from fresh fruits. The vasculature of the basement membrane is associated with Type IV collage and needs to be hydroxlated. If you do not do anything about this, then you will die. 19 – Curing Scurvy  They used to think it was the bad air once you crossed the warm climates that caused scurvy. Limes stopped scurvy. 20 – Denaturation of collagen containing a normal content of H0-proline and abnormal collagen containing no HO-proline  HO-Pro residues are important for the stability of the triple helix. This is the melting curve of a triple helix. Normal collagen is stable beyond our body temperature. Resistance to denaturation is around 40°C. Without HO-proline H-bonds, the triple helix is stable above 20°C and you lose about 50% helix content. This emphasizes the importance of hydroxylation. The most fundamental function of hydroxylation is to maintain stability of the triple helix. Is it only HO-proline residues that are important? No, Gly residues are also important. What are the temperature requirements?  What does it mean it takes a couple months for you to start showing signs of scurvy? This means that the matrix, collagens, are stable for the most part, but they keep turning over. You are remodelling the entire matrix. You start to fall apart because in matrix remodelling, you are not making proper collagens, and it all falls apart. What does it emphasize about matrix turnover with respect to scurvy? That it isn’t a permanent state. You make collagens but it is not permanent. 21 – Inhibition of collagen secretion with α,α′-dipyridyl  You can look at the impact of hydroxylation by using α,α’-dipyridyl, which blocks hydroxylation but has no impact on protein synthesis. They used radioisotopes so that you can follow the synthesis of protein in tissues. They chose C-proline is an abundant amino acid in collagens. Hydroxylation leads to decreased triple helix stability and therefore collagen secretion.  ER quality control is an important event for protecting our cells if one of your genes for matrix molecules is defective, which you inherited from one of the parents. I don’t want you to remember the molecular structure of this compound. I want you to remember its use. You don’t have protein synthesis affect. It is hydroxylation. 22 – Glycosylation  There are glucose and galatose residues added onto the triple helix. The role of those residues is not totally understood, but recently it is found that turnover of matrix molecules may be promoted by the sugar residues that are coating the collagen. How do you internalize a collagen fibril? Macrophages seem to be able to go around a particle that is much larger than the plasma membrane and internalize the particle. Fibrils are out of range for any macrophage. You end up with some cleavage with metalloproteinases which are specific for the ECM that can break down matrix. As a result, you can internalize the partially broken down matrix to go to the lysosomes and finish the job. You don’t totally break down the matrix. This glycosylation seems to be important for that. 23 – Disulfide bond formation and molecular chaperones  There is a whole bunch of molecular chaperones. We talked about the prolyl cis-trans isomerase as a chaperone in the ER that helps orient the proline residues in the trans state to allow the chain to form triple helix. We talked about hydroxylases which will stabilize the helix. There are BiP chaperones. You have a PDI, which is a key one because before you align them together, you have to make sure that they are in register so when you form the triple helix, they are all in one place. The formation of disulfide bridges in the C-terminal end help register the α-chains in space so that they will form a proper helix. This is an important enzyme that I want you to remember.  What does a PDI do? It helps form disulfide bonds. If it misses a disulfide bond, then it can correct it. It has the ability to proofread.  You start from the C-terminal end, but you need the cooperation of several key events in the ER that ensures that this occurs properly. 24 – Extracellular processing of Type I collagen  We are looking at the pro-collagen molecule. The pro-domains are targeted by two specific enzymes. There is an N-peptidase which is a member of the ADAMTS family. Another major subclass of MMP is called the ADAMTS family. A disintegrin differentiates ADAMTS from MMP. Integrins are responsible for interacting with ECM molecules. Disintegrins has this sequence that likes to bind to integrin and sterically interferes with its interaction with matrix molecules.  These two enzymes, ADAMTS-2 and ADAMTS-3, cleave the N-terminal. BMP1 is not bone morphogenic protein. BMP1 is an MMP. BMP1 removes the pro-sequences at the plasma membrane surface. They are not intracellular enzymes, otherwise if they were added in the ER, you will remove the ends at the membrane.  You must remove the C-terminal pro-peptide for any fibril to form. There are examples where the flexibility of the collagen fibril is enhanced by not completely cleaving the N- terminal pro-peptide. No polymerization can occur if you don’t cleave the C-terminal pro-peptide. 25 – Tropocollagen  I drew this plasma membrane to emphasize an extracellular compartment. This would be inside the cell. This would be the plasma membrane. This would be a tropocollagen. When the N- and C- pro-peptides are removed, it is called a tropocollagen molecule. Removing the sequences will change the solubility of the tropocollagen molecule such that it can self-assemble into fibrils.  The telopeptides are the non-helical component of the tropocollagen molecule. They are special because if molecules are going to be assembling together and they create a
More Less

Related notes for CSB327H1

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.