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Biochemistry of Collagen

by Kevin Ahern, PhD
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    00:01 Now, the strands of collagen are made originally in the endoplasmic reticulum of cells.

    00:07 That’s where the synthesis of collagen begins. Within the endoplasmic reticulum, the hydroxylation of proline and lysine side chains that occurs and this is critical both for the release of the collagen from the cell and also in providing the strength that’s necessary for the collagen to support all the different things that we described. The hydroxylation that occurs on these two amino acids is necessary as I said for secretion. If something happens at hydroxylation and prevents it from happening then the collagen chain will not be released from the cell.

    00:40 The secreted collagen chains form a helix spontaneously on their own as they are being released.

    00:47 The helices, as I said, cross-link for strength, meaning that they form chemical bonds between each other.

    00:53 Now, this slide shows the amino acid sequence of a portion of one of the chains. This is what we call the primary structures we’ve described earlier and other things about protein. It contains the sequence of amino acids. As we look at this sequence, we can see some interesting things.

    01:10 There are three amino acids that jump out, the first of those being hydroxyproline.

    01:16 Hydroxyproline is shown as the Hpr in this diagram. Hydroxyproline is not a natural amino acid.

    01:23 It’s formed by the chemical modification of proline which occurs after the collagen has been made.

    01:29 It’s called a post-translational modification. Hydroxyproline doesn’t occur in too many proteins.

    01:35 Proline, we also noticed is very abundant within this region of the collagen primary chain.

    01:42 This abundance of proline is particularly unusual for a helical type protein because proline in most proteins forms bends and bends are not consistent with a helical structure.

    01:55 The third amino acid that’s very abundant here is glycine. Now glycine, we see occurs every 3rd residue.

    02:04 It almost always occurs adjacent to a proline. That turns out to be really important because glycine has a very small side chain. Proline has a very bulky side chain.

    02:14 So, putting these two together, glycine allows the space for proline to actually be able to be within a helix. Without glycine, collagen would not have the structure that it does. Now, the other one that may not jump out at you at first but I have an arrow pointing here is that of lysine.

    02:31 Lysine occurs fairly infrequently within a given collagen chain but lysine is very important as we shall see for making the cross-links that hold the individual chains together. Now as I said, glycine occurs every 3rd residue.

    02:47 It is usually followed by a proline or in some cases by a hydroxyproline. Again, this is to allow the space for proline to exist in a helical chain as seen here. The hydroxylation of protein is critical, not only for the release from the endoplasmic reticulum, as I’ve mentioned previously, but also for allowing the overall collagen chain to be stable. This hydrogen bonding that happens between individual proline residues is very critical for keeping the collagen stable at the temperature of the human body. Without that hydroxylation, collagen will fall apart. The hydroxyproline almost always precedes a glycine as you can see again in this sequence. In this slide, we can see the hydroxylation of proline to make hydroxyproline; proline, of course, on the left and hydroxyproline on the right.

    03:39 Proline is originally put in to collagen in the form that you see on the left. The chemical modification I’m getting ready to describe to you produces the hydroxyproline on the right. Making the hydroxyl group onto proline to make hydroxyproline requires molecular oxygen. The enzyme catalyzing this reaction, as you can see in the name is a dioxygenase. Dioxygenases use molecular oxygen. They're rather unusual enzymes used in catalyzing fairly complicated reactions. In this reaction, we can see that there’s a cofactor of α-ketoglutarate that is needed to make this overall reaction occur. One of the oxygens from molecular oxygen is used to produce the hydroxyl on the hydroxyproline. Well, that requires a reduction.

    04:24 Reduction requires electrons. So, how does that overall process happen? Well, the electrons that are used to make the hydroxyproline actually come from ferrous iron, the Fe2+ that you can see above the reaction up there. The roomful of electron from Fe2+ to make Fe3+ is what makes the hydroxylation reaction possible. Loss of ferrous iron to make ferric iron, that is from 2+ to 3+ is essential for the reaction but the cell only has a limited amount of ferrous iron. If all the ferrous iron gets converted into ferric iron, then the cell is in trouble and would not be able to exist.

    05:05 That means therefore that the ferric iron, the 3+ has to be converted back to the 2+. How does that happen? Well, that turns out to be one of the most interesting and important reactions that I’m going to describe to you here. That’s because the electrons for making the ferric go back to the ferrous comes from vitamin C, ascorbate in the reaction that you can see above. Ascorbate is vitamin C. So, we need vitamin C to keep this reaction going. If we don’t have enough vitamin C, we will not be able to produce hydroxyproline.

    05:38 I’ve already told you that hydroxyproline is necessary for making stable collagen. So, this is pretty important.

    05:44 Well, we have a limited amount of ascorbate as well. So, what happens to all the ascorbate if we give up all of its electrons and make the other molecule that you can see here, the monodehydroascorbate.

    05:56 Well, it turns out that the electrons for that come from NADPH. The cell has a pretty abundant supply of NADPH.

    06:03 So because of the combination of all three of these reactions, the cell can make as much hydroxyproline as it needs. The enzyme that I showed you on the last slide was described as a dioxygenase.

    06:16 That was to emphasize the molecular oxygen that’s used in the reaction. However, this dioxygenase goes by a variety of names. Probably a better way to describe it is as a prolyl hydroxylase as seen here.

    06:28 The prolyl hydroxylase is depicted in the structure on the right. Now, prolyl hydroxylase recognizes a specific sequence that’s found within collagen. That sequence is known as x-Pro-Gly where x is any amino acid, Pro of course is proline, and Gly is a glycine. We’ve seen within the collagen sequence that proline and glycine occur fairly frequently. So, the enzyme doesn’t just randomly put hydroxyl groups onto prolines but only those that have in the proline, in the sequence like as you can see here.

    06:59 That means therefore that this hydroxylation reaction occurs in a lot of places within collagen.

    07:05 That’s very important because again, it gives collagen some stability. Now, this chemical modification that I’ve just described is the most abundant post-translational modification that occurs anywhere in the body. The enzyme is located in the endoplasmic reticulum because as I noted, this activity is essential to get the collagen exported out of the cell. Without the hydroxylation, collagen won’t be released. Even if it is released, it won’t be stable. Now, this enzyme is flexible.

    07:34 It's a little unusual about it as well. It will modify the prolines of other proteins that have the sequence x-Pro-Gly. Now, this is interesting because one of the proteins that has a fair number of x-Pro-Glys is the protein known as elastin. Elastin is as its name would suggest, a protein that gives flexibility to tissues. Another protein that gets hydroxylated by this enzyme is called HIF.

    07:58 HIF stands for hypoxia-induction factor. The hypoxia is a condition where there’s limited oxygen available for cells. HIF is activated when there’s low oxygen concentration. Well, how does the cell know when there’s low oxygen concentration? One of the ways is by the hydroxylation reaction.

    08:19 You saw that hydroxylation requires molecular oxygen. In low molecular oxygen concentrations, no hydroxylation of HIF will occur. When no hydroxylation occurs, HIF is active. On the other hand, when there’s abundant oxygen, hydroxylation of HIF causes HIF to be destroyed.

    08:39 So by modifying HIF in this way, under high oxygen conditions or no oxygen conditions, the cell can tell how much oxygen is there and whether HIF is actually needed.


    About the Lecture

    The lecture Biochemistry of Collagen by Kevin Ahern, PhD is from the course RNA and the Genetic Code. It contains the following chapters:

    • Biochemistry of Collagen
    • Hydroxylation of Proline
    • Prolyl Hydroxylase

    Included Quiz Questions

    1. It occurs in the endoplasmic reticulum.
    2. It prevents the release from the cell.
    3. It occurs on arginines and alanines.
    4. All of the answers are true.
    5. None of the answers are true.
    1. ...glycine.
    2. ...proline.
    3. ...hydroxyproline.
    4. ...lysine.
    5. ...hydroxylysine.
    1. It gives collagen more thermal stability.
    2. It occurs after export from the cell.
    3. It directly requires vitamin C.
    4. All of the answers are true.
    5. None of the answers are true.
    1. It uses molecular oxygen in its catalysis.
    2. It converts proline to hydroxylysine.
    3. It reduces iron to Fe++ in its catalysis.
    4. All of the answers are true.
    5. None of the answers are true.
    1. All of the answers are true.
    2. None of the answers are true.
    3. It is found in the endoplasmic reticulum.
    4. It catalyzes a reaction necessary for collagen export.
    5. It catalyzes the most abundant post-translational modification in the body.

    Author of lecture Biochemistry of Collagen

     Kevin Ahern, PhD

    Kevin Ahern, PhD


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