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Section 11: Extracellular Macromolecules

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1 Section 11: Extracellular Macromolecules
Fibrous proteins: keratin, collagen and elastin 02/21/06

2 Selected Extracellular and Cytoskeletal Proteins
Connective Tissue Fibrous Proteins collagen elastin keratin fibronectin Other Fibrous Proteins fibrin myosin (partially) Cytoskeleton Proteins actin intermediate filaments microtubules There are different categories of extracellular and cytoskeletal proteins. The connective tissue fibrous proteins are the main ones that we will talk about, collagen, elastin and keratin. Fibronectin is another example, not there in same quantity. Other fibrous proteins, fibrin, we had for blood clotting. As is the case, these fibrous proteins are typically architectural, are involved in communication, but not enzymes. Myosin is an example of a partly fibrous protein, long tail of coiled coil of alpha helices is typical structure. It has an enzymatic portion. Then there are the cytoskeletal proteins, that are inside the cell and keratin, in both categories, produced in cells that make it until they are filled, cell dies, then keratin has structural and extracellular function. Intermediate filaments exist in the smooth muscle, connecting the components of it. Microtubules are reminiscent, similar in function, to actin, form long polymeric structures on which motors move, but not myosin. In an axon, which is highly asymmetric, if you counted on diffusion to get things from the nucleus, where genes are being expressed and things are synthesized near there, diffusion wouldn’t work, you would wait too long for it o have a timely effect these microtubules run the length of axons, non myosin type motors move things along the microtubules, way to distribute things within the cell 1

3 Cell Adhesion Receptors and Integrin are transmembrane proteins 2
Schematic pic of two types of cells connected to one another. Diagram shows some of the proteins involved. This is epithelial cell and fibroblast. At the center there is a layer of proteoglycans, proteins that have some number of long chain polysaccharide, or oligosaccharide attachments. On either side of that is a layer of collagen, collagen IV. Then in contact with and communicating with the collagen, there are molecules that connect this extracellular matrix to the cells. Those are proteins that have names like adhesin, and bindin. They are more strictly structural. Shown here, examples of proteins that transverse the plasma membranes, or the membranes of the cells so that it allows for communication between extraceullar matrix and interior. Its possible to have lines of communication between the cells. If we get to the specifics, there is a laminin entactin protein which bind to laminin receptor, and the receptor crosses intermembrane protein, and then there is fibronectin, which is shown here. Binds to fibronectin receptor, then that receptor crosses to the interior, to cytosol, then integrin, which crosses the cell. Receptors and Integrin are transmembrane proteins 2

4 Cell adhesion proteins
A chain B1 chain B2 chain Collagen binding © 2000 by Geoffrey M. Cooper Enactin binding Proteoglycan binding Cell binding Collagen binding Cell binding Cell binding Laminin Fibronectin Cell adhesion proteins Proteoglycan binding Actin Some of these proteins. Example of laminin, a fibrous protein, notice how many binding sites there are on this laminin. Binds cell, collagen, enactin. Its also got a proteoglycan binding site. Helps hold things together and to allow communication through plasma membrane of the cell. Fibronectin, with collagen, cell binding, proteoglycan binding, and other binding sites that are fibrin binding. One of its many jobs is to coordinate blood clot formation. Picture of integrin, in this structure the intracellular matrix is at the bottom, integrin is going across the plasma membrane, where its bound to some interior proteins, that make up the cytoskeleton. When I talked about actin, it had a specific function in force generation by interacting with myosin, but almost all cells have actin, it isn't always in these nice regular bundles, it is typically filamentous, exchanging between actin monomer and filaments, remember that double strand of globular actins with helical twist to it. In this picture it shows the actin and then some other proteins. Don’t worry about specifics, see the connection between ECM with PG and collagen bound to an integrin which then crosses the membrane so things that happen outside can be communicated mechanically, because it is physically transversing the cell, but in the case of receptors, something like laminin receptor interacts with laminin bound to intracellular matrix, face to interior of cell, communicates by conformational changes, chemically with regard to intracellular signal transduction. Integrin Plasma membrane Integrin Extracellular matrix Matrix binding 3

5 Association Between Cell and Extracellular Matrix
Some of the fibrous proteins are transmembane and connect (and communicate) to the cytoskeleton (actin, keratin, microtubule, tailin, vinculin). Fig , Lehninger. The strands are the collagen, the PG shown. Then there is fibronectin. The reason to look at this is that it shows the actin filaments which are bound to the dark globular looking, transmembrane proteins, that bind to and interact with actin, the way the integrin did previously. The actin provides structural framework within the cell, but not geometric. That’s all to say about cytoskeleton, relatively new area in biochem. 4

6 Elastin Elastin (64-66kD) is rich in prolines and non-polar side chains, and one third of its amino acids are glycine. As a result, its has uncommon secondary structure (more random structure than found in other proteins). It does not have a stable tertiary structure. Elastin is very resilient. It can be stretched to lengths many times greater than in its relaxed state. It can also be compressed. Elastin is common in many connective tissues, along with collagen, especially if the tissue undergoes physical stress. It surrounds arteries, is in the lung and in ligaments. An important extracellular protein in connective tissue around arteries, heart, lung, ligaments things which typically stretch more than other tissues might. Its very high in prolines, and very high in non polar structures so that it excludes water. About 1/3 of its amino acids are glycine, which is the one with the smallest side chain, just a hydrogen. For many years it was thought that it had no secondary structure, a random structure. Turns out that’s not true. Beta spiral. The structural property of elastin is that it is elastic. And by that it is resilient, if you stretch it, returns to original conformation, if you squash it, it returns to original structure. This is why it is a structural component for things that will be flexible like the lung where shape of compartments are changing all the time, and elastin structure makes up architecture of those. Also occurs in joints, to push in pressure that occurs on joints, and occurs in ligaments. The matrix metalloproteinases that degrade proteins in some cases as part of the response to inflammation, and in other cases as a result of invading species, bacteria that has metalloproteinase, degrades the elastin and that increases inflammation and allows bacteria to invade tissues. 5

7 Elastin Structure and Function
Elastin interconverts between a number of conformations, both disordered (upper two on left) and b-spiral (bottom left). After cross-linking, when elastin is stretched (or compressed) it is less stable and it returns to the disordered conformations. Beta spiral. It has hydrogen bonds, transient, but compared to alpha helix or beta sheet it is secondary structure. If this is relaxed, and this is an extended chain, this does not represent alpha helix, there is cross linking holding the chains together. If you stretch them, elongated form of individual elastin monomers, held together by cross link. If you release whatever stress created the elongated structure, it spontaneously goes back to relaxed structure, does this in order to maximize the entropy. Example of entropy driven reaction. You would have to put energy in, to get it to elongate, this is a measure of disorder, so this is a more ordered structure, because they are elongated and lined up. When you take away the stress that is holding it in this structure, goes back to optimize entropy. If you were to squash it, it can be made smaller, and it would take energy to do that, then when you release pressure, more ordered because in smaller volume, release and it would go back to relaxed state that is larger. This is reversible. Resilient, flexible. (Fig. 4-28, Rawn) (Fig. 4-30, Rawn) 6

8 Elastin Cross-linking
Some lysine residues in elastin are deaminated and oxidized to the aldehyde level. They combine with each other and with other lysines to form lysinonorleucine and desmosine cross-links Cross linking derived from lysines. Two primary structures. Lyinonorleucine, two lycines contribute, one elastin a lysine in one elastin polymer and what was a lysine in the other. One of them has been oxidized and deaminated. This is derived from two lysines where one loses its amino group, form covalent linkage. Linkage between elastins is as strong as covalent peptide bonds. More complex structure, desmosin is made up of four contributing side chains, each was a lysine, only one has the nitrogens and the other is oxidized to aldehyde from, and forms cross linking reactions. Cartoon doesn’t show four connected but you can 7

9 Keratin Keratin is rich in cysteines.
Fig. 3.34 Keratin is rich in cysteines. Its secondary structure is mostly a-helical. The helices form coiled coils (on right). The coiled coils pack into higher order elongated structures. Keratin properties depend strongly on the degree of disulfide cross-linking. With low levels of cross-linking, it is flexible (hair, skin). It can be made very hard with additional cross-linking (claws, horns). Extracellular via whithering of keratin-filled cells. Intracellular: cytoskeletal intermediate filaments. 2 nm This lecture from structure function point of view, understand why some function occurs as a result of the structure of the molecules. The three examples, elastin, keratin, and collagen, have macroscopic properties that are directly related to the actual molecular structure. In the case of elastin, that structure was this spiral, random, coil type of structure, stretch and it springs back. Keratin is rich in cystines, almost all alpha helical. It forms coiled coils. this is similar to the tail region of myosin, two alpha helices that coil around one another. Coiled coils pack into more elaborate structures. There is cross linking, disulfide, between the chains of a single keratin structure and also between neighboring keratin structures that exist in higher order. Keratin is the main constituent of hair and nails, as well as the nails, there is a wide variation in how hard those different materials are. Keratin is also in skin, skin and hair are flexible, soft, claws and horns are not. The way that hardness is modulated is degree of cross linking. More cross links, harder the material. It results from cells being packed with keratin, cell when it is still alive makes keratin until its filled and then it dies but the cell stays in place filled with keratin connected to other cells. This structure is also occurring in other cases, like intracellular intermediate filament. 8

10 Keratin Supramolecular Structure
Two coiled coils bind together to form a protofibril (below). Protofibrils assemble into various microfibrils (on the right). ¬Fig. 4-5 Fig. 4-6® Rawn The keratins form a protofibril when two of them form a complex that has a helical. Cross section is the two strands of alpha helix that make up keratin, binding to one another to form protofibril, four individual alpha helices, cross section of protofibril, four pink and orange circles. Protofibrils further aggregate to microfibrils. Two major microfibril arrangements are the square and the hexagonal, round shape. They are hollow inside, cross linking is along the length of the protofibril, everything is cross linked, with some there is enough resilience to regain its original structure. If you have a long hair and you can take some of it and pull on it, if you don’t pull too hard it stretches then goes back to original shape. Stretching alpha helices and when you let go it goes back to alpha helical form, guided back to that structure by sulfhydril cross linking. More cross linking, can bend finger nails. 9

11 Keratin Cross-linking
The structure of keratin is strengthened by disulfide cross-links from one helix to another. Cross linking is disulfide cross link. You can make and break these cross links in the case of hair in reversible fashion. You can do that by manipulating this reaction, one method is to use heat, get the hair in the shape you want. Idea that you get it in that shape, heat it up, break disulfide bonds, then they reform and it maintains the structure for a while. Only form some of them. Another way to do it is chemically, chemicals break the bonds then other chemicals to form them, called a perm. Too much heat or chemical and it falls out. These are the keratin cross links. 10

12 Collagen Types Fibrils – long triple helices
I. Skin, tendon, bone, dentin II. Cartilage and vitreous humor III. Skin, muscles, blood vessels (frequently found with type I) V. Fetal tissues, placenta, interstitial tissues XI. Cartilage Fibril associated – interrupted triple helices IX. Cartilage, vitreous, humor XII. Embryonic skin and tendons XIV. Fetal skin and tendons Fibril associated -- beaded VI. Most interstitial tissues Sheets IV. All basal laminae VIII. Endothelial cells, X Cartilage growth plate Collagen is the protein that is present in the largest amount in the animal world. There are many different varieties of it, categorized, collagen type refers to the numbers, don’t go in order, grouped by their structures. Collagen I II III V and XI have long triple helical structures. They appear in different places, collagen I appears in bone and dentin and tendons. Collagen IV is the one depicted in the intercellular matrix, sheet like structure. There is a subcategory of the long triple helix, these interrupted triple helices, triple helix then part is not. the point is that some of these are constituents of things important to you. In dentin, bone and basal lamina, matrix between cells are particular types of collagen structure. 11

13 Collagen Collagen has glycine in every third position, is rich in proline, and contains hydroxyproline and hydroxylysine residues. Collagen does not have secondary structure, but three highly extended strands interact to form a triple helix. Collagen triple helices form a rod-like fibril or sheet aggregate that is somewhat flexible, not extensible, and can be very strong. Cross-linking increases its strength. It is common to connective tissue and is present in bone, dentin and cementum. Collagen is the most abundant protein in the biosphere. Collagen is also 1/3 glycine the way elastin is, but in this case its in every third position, A B glycine. It also has extensive post translational modification of lysines and prolines which are hydrolxyalted. That is the reaction that requires vitamin C. Forms a triple helix, collagen helix, and its cross linked, changes the degree of strength. Collagen in the skin is not as cross linked as in Achilles tendon. Cross linking gives strength, but never as hard as keratin. The structure is very elongated, unlike the elastin or keratin structures, if you pull on it, molecular ability to extend, collagen is already extended, good connective for things like bone to muscle or bone to bone. Its also present in bone and cementum. Its present in formation of enamel, but none left in mature teeth. 12

14 Collagen Triple Helix Prolines, especially hydroxylated prolines, keep the individual chains extended, and increase Tm (keep it above body temperature). The small size of the glycine sidechain in every third position allows the three strands to come close together. There are interstrand hydrogen bonds. It is not very extensible because it is already extended (3.1 D per residue vs 1.5 D for a-helix). Glycosylation of hydroxylysines appear to modulate fibril or sheet formation by the triple helices. Triple helix, side chains not shown. The individual beads that are connected by rods, idea is that each sphere is an amino acid, and the chains are extended. The prolines, which cannot for alpha helix, contribute to stability by hydroxylating prolines, contributes more with having the elongated structure. Glycines by being in every third position provide minimum bulk in the interior of this structure. The point at which the strands have the side chains facing one another, has a glycine every time. That minimizes the interior bulk and allows three strands close to one another. Hydrogen binding between the strands. Not extensible, already stretched out. There is 3.1 angstroms per residue, compared to 1.5 for alpha helix. Double the length of alpha helix before you get to a structure as extended as this. The lysines are hydroxylated then glycosylated, with disaccharide of glucose and galactose. Degree of glycosylation of these side chains determine when you aggregate triple helices if you get bundles or sheets. Fig. 11-5 Stryer 3rd 13

15 Collagen Amino Acid Modifications
The hydroxylation reactions. Start with protein, this is part of the polymer. The protein is hydroxylated, likewise here is lysine part of collagen that is also hydroxylated. Carbon gets hydroxyl group,, glycosylated. The reaction is catalyzed by prolyl hydroxylase, uses molecular oxygen and alpha keto glutarate as a coreactant. Decarboxylated, put on hydroxyl group, inactivates the enzyme, left with oxygen bound to iron III in active site, that is reduced to give active form of enzyme by oxidizing ascorbate. This is the reaction that keeps one from having scurvy, if you have enough vitamin C. I9f you don’t then you cant hydroxylate, don’t make good collagen, periodontal ligament is weak, teeth move around, bleeding gums. Not a coenzyme but required to keep enzyme in active form Hydroxyprolines form interstrand hydrogen bonds. These post-translational modifications require ascorbate to reverse the prolyl hydroxylase active site Fe(III)-O- . 14

16 Glycosylation of Hydroxylysine
Once lysines are hydroxylated, galactose and glucose added. This is the usual way monosacc are added to things. UDP activates them. The more of this there is the more likely you get sheet like structures as opposed to cable like structures. The hydroxylysines are modified by sequential glycosylations, giving lysyl-gal-(a1®2)-glc. Activated sugar complexes are usually UDP-sugars. Higher levels of glycosylation favor formation of sheet structures by the collagen triple helices. 15

17 Procollagen and Tropocollagen Formation
Fig. 4-24 Rawn there is aggregation of these, the way keratin aggregates into protofibrins. But first, production of procollagen. In the cell before secreted, there is a portion of the individual strands, so that the depiction is intended to represent extended strand. These are like the three strands from the beginning, yellow red and blue, but now there is this additional portion at C terminal that has disulfide links. This allows them to get close enough together to form the energetically unfavorable long extended structure. This keeps them together, do form the triple helix, assembles into procollagen, once this structure has formed, then procollagen peptidase removes the portion at the end, tropocollagen molecule. Start with procollagen, has portions required for assembly into triple helix, remove them proteolytically, end up with tropocollagen The three strands of procollagen are cross-linked by disulfide bonds near the C-terminal end, initiating triple helix formation. Procollagen is soluble, but tropocollagen, formed by hydrolyzing away peptide fragments at both ends, is not. 16

18 Aggregation and Cross-linking
Tropocollagen spontaneously aggregates into elongated staggered arrays, shown in two dimensions at right. Hydroxylysine glycosylation determines fibril or sheet formation. Cross-linking strengthens the structure (lower). In bone, dentin and cementum, biomineralization begins in the gaps (hole zones) between the individual tropocollagens (type I). Fig. 4-24, Rawn Tropocollagen spontaneously aggregates into structures that are shown here. If there is staggered array, and space between the tropocollagen from right to left. They look like this on EM. Some overlap vertically. Cross linking gives strength. Variable degrees of cross liking depending on function of collagen. Once they are cross linked, this is an example of type I collagen in bond dentin and cementum. By a mechanism that is not fully understood, at least the location is identified the biomineralization occurs in gaps between tropocollagen and collagen structures. What appears as holes, will remain as holes, that is a nucleation center for biomineralization. That is where it starts, then as mineralization proceeds you get degradation of collagen to various degrees depending on bone, cementum, enamel 17

19 Collagen Cross-linking
Details of cross linking. Lysines are contributors. They can be oxidizes to aldehyde level, covalent bond, non enzymatic, spontaneous. If you have a lysine, similar to desmosine, only one nitrogen in the complex, only one lysine intact, the other has been oxidized to aldehyde. If you have one of these and two of these nearby spontaneously cross link. Cross linking depends on how many lysines there are and how activate the lysyl oxidase Lysines and hydroxylysines are used in cross-linking. Other than oxidation to the aldehyde level, the reactions appear to be non-enzymatic. 18

20 Fibroblast to Mature Collagen Fiber
Fibroblast cell is the box. First polypeptide synthesis, then the post translational modification, modify the amino acids. Hydroxylations and glycosylations. Triple helix formation occurs and we have procollagen formation. That is secreted into extracellular matrix in a vesicle via exocytosis. Procollagen in the vesicles, once its outside get proteolysis that turns it into tropocollagen that spontaneously aggregates. Similar to fibrin story, not occurring in the blood. Procollagen is like fibrinogen, and tropocollagen is like fibrin before its cross linked and is affected by proteolytic enzymatic action. These tropocollagens assemble into collagen fibers, then cross linked for strength, occurs in extracellular matrix where you make ECM or tendon or other structure. Procollagen in vesicles is transported to the cell membrane in vesicles, and then secreted via exocytosis. Proteolysis, assembly, and lysine oxidation leading to cross-linking occurs outside the cell. 19

21 Collagen Degradation Collagenase cuts the 1000 aa triple helix into 250 and 750 aa fragments which melt and are proteolyzed. Some animal tissues (for example tadpole tails) have collagenases that are used to degrade collagen during growth and remodeling. The collagenase of Clostridium histolyticum destroys host connective tissue, helping to make it a highly invasive bacterium. In periodontal disease, host collagenases help break down periodontal ligament (collagens type I and III). Turnover. EC proteins are not bathed in the enzymes that produce them. As a result, when you do damage to tendons and ligaments, healing is slow. Degradation of when its done purpously, collagenase cuts the large triple helical structures into smaller, when they are smaller don’t maintain extended triple helical structure, melt, don’t maintain a triple helix, that makes them susceptible to proteolytic enzymes collagenases that break them into smaller pieces and eventually into amino acids. Some cases this is controlled in a positive sense, when tad polls lose their tails, collagen in the tail, collagenases secreted there, reabsorbs it. Doesn’t fall off, absorbed back. There are also bacteria that use collagenases to invade. Break down host collagen, allows bacteria to be invasive. It has a very effective collagenase that makes it invasive bacteria. In the case of periodontal disease, often host collagenase which is activated, produced in greater amount because of inflammation, response to it, host collagenase begins degrading host collagen, contributing to the damage done by periodontal disease 20

22 Section 12: Mineralized tissues. Calcium and phosphate metabolism
Next topic: Section 12: Mineralized tissues. Calcium and phosphate metabolism

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