Fundamentals Dr. Miller Part 2 11:00-12:00

Fundamentals Dr. Miller Part 2 11:00-12:00

After Break

Slide 11

·  Collagen is made through polyribosomal protein synthesis like most proteins that we make go through this system.

·  Collagen chains are put in the ER. They come together and the associate by virtue of the interactive capabilities of the C-terminal non-helical globular domain.

·  In other words, that procollagen piece at the C-terminus has an assembly function and it allows three chains to come together and the formation of the helix is spontaneous which is exergonic reaction (gives of energy; spontaneous).

·  You wind up with a molecule that is fully functional and asymmetric with N-terminal golubular domain and same C-terminal globular domain (pervious slide).

·  Notice that the hydroxylation occurs on the proline and lysine side chains.

·  Glycolasation appears on the hydroxyglycine chains during synthesis. These are co-translation modification, not post-translation (says that in the book). While the chains being synthesized, the hydroxylation occurs.

·  A post-translation modification would be cleaving pro-chains away. Prolyl hydroxylation is more important the glycoslation.

o  The proyl hydroxylation gives more opportunies to h-bond formation and stabilization of the helix. If you don’t have this hydroxylation, then you can not form a stable helix and you can not secret the molecule.

·  The molecule must be in native before it can go the golgi and to the Extracellular space. This conformation must be achieved before it secreted.

·  If it can not be secreted, then you will be deficient in collagen. One way is by lack of enough Vitamin C.

o  Vitamin C major function is be a co-factor in the hydroxylation of proline residues. If it does not occur, you can not get perfect collagen formation.

o  This leads to Scruvy: gives to bleeding joints, gingival (gums). Any kind of tissue that is constantly damage d and needs to repaired like joints (knee, ankle, TMJ) with new collagen. They start to break down if Vitamin C is no there. You can’t synthesize or secret native collagen molecule.

Slide 12

Vitaman C is absorbic acid, it has a pKa=4.2. It is capable of from the reduce (left side of figure) from to the fully oxidized form (right side) . IT has given up 2 protons and 2 electrons to form oxidized asboric acid.

Absrobic acid is in this reaction to reduce ferrous iron (during the reaction it has become ferric acid).

Basically the bottom reaction in words:

·  This polypeptide chain with proline. It is going be hydroxlated in the fourth position. At the same time, alpha-keto=gluterate is going to be decarboxylated to give succinic acid. Use a molecule of oxygen to do this. One oxygen molecule to goes to proline, other oxygen goes to make the carboxyl group whole again.

·  Proyl hydroxal (dioxygenase) is used. It uses both atoms of the oxygen molecules. This very important process. Keep in mind hydrolxation happens 100-125X times in a chain.

Goes back to slide 12.

·  Emphasized that hydroxylation occurs 100-125 times (hydroxlate proline )in a chain.

·  The helical region of each collagen chain has 1050 amino acids. This is large molecule.

Slide 13- Lysyl Hdroxylation

·  Hydroxylysine is made the same way by an enzyme called lysl hydroxlase. The hydroxyl group is added to the delta carbon on the side chain of lysine.

·  It is glycoslated by the short disaccharide (glactose, fructose ..etc mentioned that we won’t dwell on it. ). This occurs infrequently with collagen that forms fiber. Remember: that your place hydroxyl groups on lysine.

·  It is important for formation of stable cross-links between collagen molecules.

Slide 14. Specificity of Chain Association

·  We talked about type 1 and type 3 collagen. These have different types have different chains (from previous slides).

·  There is way to make sure that fibroblast keeps them separated.

·  Fibroblast makes these chains: alpha 1 (type 1), alpha 2 (type 1), alpha 1(type 3) . These are synthesized at the same time in the cell.

·  The carboxyl domains that are responsible for association.

·  For example type 1 : has two alpha 1 chains, and one alpha 2 chain. That type of ratio is maintained by the inherent of operation of the C-terminal globular domain. It will be removed eventually. To get the right chains together, the C-terminal domain controls it

·  If you isolate collagen from skin, you can separate the types of collagen. You will find the correct ratio of two alpha 1 chains, and one alpha 2 chain. You will never mix them up (mixing of the chain types).

Slide 15- Chemical Stability

·  When the helix is formed, it is formed into a coiled coil structure.

·  Secondary structure - polyproline helix

·  Tertiary structure- 3 polyproline chains wrapped around each other to make triple helix.

·  Minor helix – polyproline helix; left handed

·  Major helix- The coiled chains; right handed

This type of arrangement (like in other polypeptide chains) causes to be erroneous.

·  When you have native collagen with a conformation like this, typsin, chyotrypsin, cleostripin (protelytic enzymes) are worthless.

o  These will not attack collagen molecules with in triple helix form. You don’t want them to attack structural molecules (places like GI tract, joints, lungs).

o  If this was the case, we would dissolve ourselves. You have structure molecules to be stable.

o  Chemical stability comes from this triple helical structure. Collagen molecules are stable.

·  Over those years of development, you have lost a great deal of connective tissue and replaced it. You get rid of the collagen molecule as an infant, and replaced with new collagen molecules.

o  That is done with collagenase- breaks down collagen. Though collagen molecules are stable, you can remove them.

o  However, if it is disorganized then you run into pathology. As an adult you don’t have collagenase running around crazy.

o  You can get disease like rheumatoid arthritis and preiodental diseases.

Slide 16 Secretions of Collagen Molecules

Secretion of the molecules goes when the molecules is confirmed completely.

-small globular domain C-terminus, and the non-helical N-terminus.

-The molecule can now be secreted to golgi apparatus and through the packets that are deliver to the extracellular matrix.

-Points out parts of the cell on the slide.

Slide 17 Extracellular Processing of Collagen

·  Once outside the cell, the extracellular process or post-translation modification f the collagen molecule.

·  The procollagen N protease and the procollagen C protease remove the globular C-terminal and the globular N-terminal. Can remove a little of the helical part. Then it’s in fiber formation.

o  There are small regions of the globular regions are left for the cross linking formation.

o  The fibers are formed and Lysol oxidase will convert hydroxylysine or lysine residues in the globular domains at the end ( called telopeptides=end peptides) of collagen molecules .

o  The telopeptides are responsible, though tiny (1% of the molecule) crosslinking.

·  To start the cross-linking , you use the fiber as the substrate and Lysol oxidase will convert the side chains of lysine, hydroxlysine to aldehyde form.

o  The cross linking can occur from this regions and other regions.

o  This will give you a quaternary structure that is made up of staggered molecules. They are linked side by side. This is the final act in making collagen.

·  You have chemically stable in the middle, now its physical stable because every molecule is linked to its neighbor (sometimes more).

Tangent:

·  If you put tension on ligament, it will not unravel because all the indiviaual part are held together. If not held together covalent, then it would be fall apart.

·  If you put extreme tension on a ligament, you can rip it apart.

·  ACL is usually torn away with football players ending their career. Tom Glavin (Braves) had a slight tendon error in the pitching. Did not need Tommy John surgery, which is when you replace the entire tendon. You have enough time for the tendon to anneal itself to the bone and the muscle. . Sometimes it takes 2 years for a tendon to seat itself back to the muscle. Hard to get tendon to get the same amount of strength.

·  Drew a picture of tendon between ulna and muscle. You have to give time to attach itself again.

Main Point: You can put enough pressure to tear them apart. Ordinary day you should not encounter that much pressure.

Slide 18- Fiber Architecture

Now you have substance that is chemically and physically stable. Theoretically it should last all a lifetime- Ideal.

·  The fibers are organized in such a way that the molecules have 1/4 stagger relative to each other along the axial axis. The number 2 is staggered by 1 D from number 1.

o  The 3 is 1D from 2, 2 D from one fiber…so forth.

·  The fifth position occupied by which is staggered by 4 D units from fiber 1. The molecules, however it is 4.4 D molecules long. There is an overlap. The 1’ is also 1D overlap from the fifth fiber.

·  That gives a gap region of .6 between Molecule 1 and Molecule 1’ . This gives the alternating (staggered) region in collagen fiber. This is why it looks striated in the EM. When you stain positively or negatively.

Slide 20 Preparation of Cross-Linking

Really look at the reaction

·  The sides chains of hydroxllysine and lysine are oxidativitly deaminated by the enzyme lysl oxidase.

o  It requires copper and molecules oxygen. You replace amino group (primary) at aldhyde function.

o  You do the same thing with lysine, you get an aldheyde function.

o  We call this hydroxylallysine (lysine) and allysine(from lysine). Remember these names.

o  These are aldehydes s are going to initiate a physiological tanning process. This process is controlled and discreet than treating with formaldehyde.

o  This reactions occurs without changing side chains of lysine or hydroxylysine.

The aldehydes can reaction with each other and make aldol condensation reaction continue to the next slide- First reaction

Slide 21

Part 2 of reaction

·  The aldhyde reacting with a primary amine on another hydroxlysine to give you dehydrohydroxylsinonorleucine ( you don’t have to remember this). This gives you product gives you schiff’s base from earlier.

·  (C)-reaction . If the aldehyde is donating from the side chain of hydroxlysine, you will Schiff base but this time you get it beta to the hdyroxl groupl

·  The hydroxyl group will oxidize and will become a ketone group. The Schiff base is reduce to secondary amine, which is stable.

·  The stable linking comes from hydroxallysine with lysine or hydroxlysine to give you hydroxylysino-5-oxonorleucine. The nor leucine (4-carbon) is the side chain. These cross-links are stable and that link the molecules together.

Slide 22- Location of Cross-Links

·  Top figure - The cross linking can be visualized as starting from the N-terminus (non-helical) region one molecule to region which is almost at the end of C-terminus of another molecule.

·  Also, the C-terminus from one molecule to the N-terminus to another molecule.

·  This is the ¼ staggered position. Every molecule can be linked to other two other molecule by two crosslinks starting at aldehydes. He shows this using the figure.

Slide 23- Cross Link in a Fiber

·  One fiber is linked head to tail with one molecule, and tail to head with another molecules.

·  In the entire fiber, each molecule is attached two other molecules at the position shown. C terminus going to N-terminus, and vice versa. If you wrap the fiber the dots would crosslink.

·  Physical stability can be generated this way.

·  This is a tanning process ( you convert skin to leather). You cross-link really with chromium salts to hit the collagen molecules to stick together.

o  If it’s smooth skin, you get fine leather. If you get old skin, then you get leather used for soles. This is tanning process, but a physiological tanning process.

Slide 24- Fibrous Collagen Summary

This is a summary slide. You go from individual amino acids from the primary structure. This leads to the triple helix gives you molecules. The molecules are tied together and get the ultimate fiber.

Slide 25- Heterotypic Fibers

·  In the cornea, you have type 1 and type 5 fibers.

o  The type 1 molecules come in the fiber completely not having procollagen parts.

o  The type 5 fibers still coming with N-terminal non-helical end dangling. This has to stick out on the surface of the fiber.

o  That restricts the formation of the addition of new molecules and keeps the fiber rather thin.

Slide 26- Mineralization

When you have dentin, bone structures made, you have the collagen fibers and the gap regions between the molecules in the fibers is filled with hydroxappatite crystals. This is what gives you the calcified structure. (dentin and bones are chemically equivalent in this way)

Slide 27 Alternate Aggregates

Figure 1

·  These molecules are the non-fiber forming collagen. This is type is where the molecules join head to head.

·  This anti-parallel arrangement and these molecules join together through the non-helical region of the Carboxyl terminus.

·  These molecules are flexible because they don’t have a triple helix not all way through. The combine together these hexagonal structures.

·  The hexagonal structure will be cross linked through their N-terminus regions that dangling out.

Figure 2

·  This fine structure of a basement membrane. This is designed to be a selective filter.

·  For example, if you have a large collagen fiber (bone/dentin), you will not need a filter because the fibers will not allow them to pass.

·  In the glomerus basement membrane, small molecules like urea, calcium, phosphate should pass through and need to go into the urinary tract. Large molecules like immunoglobin and albumin must not pass through, that’s why the meshwork is needed (selective filter).

Figure 3

·  Simple fiber formed by aggregates of two molecules joined at the amino terminus ends. This is type 7 collagen.