BIOCHEMISTRY OF MUSCLES, MUSCLE CONTRACTION. BIOCHEMISTRY OF CONNECTIVE TISSUE
It is classified as: skeletal, cardiac, or smooth muscle.
Function of muscle is to produce force and cause motion, either locomotion or movement within internal organs. Much of muscle contraction occurs without consciousthought and is necessary for survival, like the contraction of the heart, or peristalsis (which pushes food through the digestive system). Voluntary muscle contraction is used to move the body, and can be finely controlled, like movements of the eye, or gross movements like the quadriceps muscle of the thigh.
There are two broad types of voluntary muscle fibers, slow twitch and fast twitch. Slow twitch fibers contract for long periods of time but with little force while fast twitch fibers contract quickly and powerfully but fatigue very rapidly.
The muscular system includes three types of muscles. They are smooth, which are found on the walls of internal organs, cardiac, which is found only in the heart, and skeletal muscles, which help strenthen the body and connect to bones.
There are three types of muscle:
Skeletal muscle or "voluntary muscle" is anchored by tendons to bone and is used to affect skeletal movement such as locomotion and in maintaining posture. Though this postural control is generally maintained as a subconscious reflex, the muscles responsible react to conscious control like non-postural muscles. An average adult male is made up of 40-50% of skeletal muscle and an average adult female is made up of 30-40%.
Skeletal muscle is a type of striated muscle, which usually attaches to tendons. Skeletal muscles are used to create movement, by applying force to bones and joints viacontraction. They generally contract voluntarily (via somatic nerve stimulation), although they can contract involuntarily through reflexes. The whole muscle is wrapped in a special type of connective tissue, epimysium.
Muscle cells (also called muscle fibers) are cylindrical, and are multinucleated (in vertebrates and insects). The nuclei of these muscles are located in the peripheral aspect of the cell, just under the plasma membrane, which vacates the central part of the muscle fiber for myofibrils. (Conversely, when the nucleus is located in the center it is considered a pathologic condition known as centronuclear myopathy.) Skeletal muscles have one end (the "origin") attached to a bone closer to the centre of the body's axis and the other end (the "insertion") is attached across a joint to another bone further from the body's axis. The bones rotate about the joint and move relative to one another by contraction of the muscle (lifting of the upper arm in the case of the origin and insertion described here).
Skeletal muscle cells are stimulated by acetylcholine, which is released at neuromuscular junctions by motor neurons. Once the cells are "excited", their sarcoplasmic reticulum will release ionic calcium (Ca2+) which interacts with the myofibrils to induce muscular contraction (via the sliding filament mechanism). This process also requires adenosine triphosphate (ATP). The ATP is produced by metabolizing creatine phosphate and glucose (stored as glycogen or absorbed from blood) within the muscle cells by mitochondria, as well as by metabolizing fatty acids obtained from the blood and within the cell. Each motor neuron activates a group of muscle cells, and collectively the neurons and muscle cells are known as motor units. When more strength is required than can be obtained from a single motor unit, more units will be stimulated; this is known as motor unit recruitment. This is spatial summation. If more strength is required than can be obtained from the current number of motor units, the motor neurons continue to recruit more motor units. When all the motor units are recruited, there will be no further increase in contraction strength. To increase the force of contraction, it is necessary to increase the frequency of neuronalT firing. This results in tetanic contraction, which is a smooth contraction. This is temporal summation...
· Smooth muscle or "involuntary muscle" is found within the walls of organs and structures such as the esophagus, stomach, intestines, bronchi, uterus, urethra, bladder, and blood vessels, and unlike skeletal muscle, smooth muscle is not under conscious control.
Smooth muscle is a type of non-striated muscle, found within the tunica media layer of large and small arteries and veins, the bladder, uterus, male and female reproductive tracts,gastrointestinal tract
, respiratory tract, the ciliary muscle, and iris of the eye. The glomeruli of the kidneys contain a smooth muscle-like cell called the mesangial cell. Smooth muscle is fundamentally different from skeletal muscle and cardiac muscle in terms of structure, function, excitation-contraction coupling, and mechanism of contraction.
To maintain organ dimensions against forces, cells are fastened to one another by adherens junctions. As a consequence, cells are mechanically coupled to one another such that contraction of one cell invokes some degree of contraction in an adjoining cell. Gap junctions couple adjacent cells chemically and electrically, facilitating the spread of chemicals (e.g., calcium) or action potentials between smooth muscle cells. Smooth muscle may contract spontaneously (via ionic channel dynamic or Cajal pacemaker cells) or be induced by a number of physiochemical agents (e.g., hormones, drugs, neurotransmitters - particularly from the autonomic nervous system), and also mechanical stimulation (such as stretch).
Smooth muscles have been divided into "single unit" and "multi-unit" or into "phasic" and "tonic" types based on the characteristics of the contractile patterns and characteristics of the smooth muscle. Multi-unit smooth muscle lines the large airways to the lungs and large blood vessels. The ciliary muscles within the eye and the arrector pili muscle of the skin are also multiunit. This smooth muscle contains few gap junctions and the autonomic nervous system innervates each smooth cell and regulates them like motor units so you can have graded responses. Single unit smooth muscle lines all the hollow organs and is most common. This type smooth muscle tends to contract rhythmically, is coupled by numerous gap junctions, and often exhibits spontaneous action potential. Another nomenclature separates smooth muscle by contractile pattern. It may contract phasically with rapid contraction and relaxation, or tonically with slow and sustained contraction. Smooth muscle in various regions of the vascular tree, the airway and lungs, kidneys, etc. is different in their expression of ionic channels, hormone receptors, cell-signaling pathways, and other proteins that determine function. Smooth muscle-containing tissue often must be stretched, so elasticity is an important attribute of smooth muscle. Smooth muscle cells may secrete a complex extracellular matrix containing collagen (predominantly types I and III), elastin, glycoproteins, and proteoglycans.
This is a specialized muscle that, while similar in some fundamental ways to smooth muscle and skeletal muscle, has a unique structure and with an ability not possessed by muscle tissue elsewhere in the body. Cardiac muscle, like other muscles, can contract, but it can also carry an action potential (i.e. conduct electricity), like the neurons that constitute nerves. Furthermore, some of the cells have the ability to generate an action potential, known as cardiac muscle automaticity.
As the muscle contracts, it propels blood into the heart and through the blood vessels of the circulatory system. For a human being, the heart beats about once a second for the entire life of the person, without any opportunity to rest (Ward 2001). It can adjust quickly to the body's needs, increasing output from five liters of blood per minute to more than 25 liters per minute (Ward 2001). The muscles that contract the heart can do so without external stimulation from hormones or nerves, and it does not fatigue or stop contracting if supplied with sufficient oxygen and nutrients.
The actions of cardiac muscle reflect on the remarkable harmony within a body and the underlying principle that individual entities in nature provide a larger function. In order for the heart to work properly, and have the necessary waves of contraction to pump blood, the cardiac cells must fire in intricate coordination with each other. In doing so, each cell provides a larger function for the sake of the body, allowing the heart to beat properly, while in turn being provided essential nutrients by the body. The coordination of the cardiac cells is essential. Should the cells fire randomly, the heart would not be able to contract in a synchronized manner and pump blood, and the body (and thus the cell) would die.
The individual actin protein is called a "globular" protein ("g-actin") because it is globular, or ball-like, in appearance. It takes many of these globular proteins coming together into a long chain to begin to make a microfilament. In fact, the actin microfilament is two of these chains of g-actin twisted up together, and the filamentous form is then called "f-actin." This is all depicted here in this schematic to the right; the two identical chains of actin are drawn in different colors only so that you can see how they come together.
You should be able to see how the microfilament can get long, so it should make sense that it runs along the long axis of the myofilament. You should also be able to see that the actin microfilament, although a doublet, is actually rather thin for its length (which would extend beyond the edges of your monitor)... that's why the actin microfilament has been nicknamed the thin filament. This actin microfilament will also be associated with some other molecules, called troponin and tropomyosin, but we won't get to that in detail until next week.
Myosin microfilaments, like actin microfilaments, are made up of many individual myosin protein molecules. However, the myosin protein is not globular. Instead, it has a head and a tail (these regions are indicated in the figure to the left). And each complete myosin molecule in muscle is actually composed of two of these head-and-tail molecules twisted around each other.
Note that there are three different polypeptides that contribute to this overall myosin protein... that is not important for you to memorize, but you will see it as you browse through the web, so I have it in this photo; you only need to know about the head and the tail for this class.
Then, to make the myosin filament, you have to take these doublet myosin molecules and put them together into large bundles. This big wad of myosin proteins is then nicknamed the thick filament. A single thick filament typically has over 200 myosin molecules in it! So it is really very thick. This is shown in the figure below (the A with the circle over it in this figure stands for Angstroms, which are a unit of length measurement... 1,000,000 Angstroms fit into one micrometer, and 1,000,000 micrometers fit into a meter; you do not have to memorize the dimensions):
Again, the thick filament runs along the long axis of the myofibril. I found the above two drawings from educational sites, but that was a couple of years ago and I have lost the links. I will attempt to find them again!
Thin filaments attach at a point called the "Z line" so that they are all lined up with one another. This is shown in this schematic to the right. The Z line is the dark line that runs perpendicular to the actin filaments. Actually, it is simply a lot of sticky proteins that anchor the actin filaments in place.
The thick filaments run in between the thin filaments. I have put them in for you to see, but first I had to change the background color of the image so that both the thick and thin filaments would be readily visible. I also had to shrink the components down a bit more.
In order to fit the thick filaments between the thin, I needed 2 sets of the thin filaments. Also, it looks like the thick filaments are just floating in the middle... however, they are anchored by proteins (of the "M line"); I just didn't show that. As you look at this image, the following items should become easier to understand:
4. If you look at the sarcomere from the side , it would look darker in the area where the myosin runs than in the area where the actin runs. Because of this, we can talk about light and dark bands. The dark band, where the myosin runs, is called the A band. The light band, where the actin runs, is called the I band.
5. How does this relate to the myofibril? A chain of sarcomeres, like the one I drew above in point #3, that runs from one end of the muscle fiber to the other end of the muscle fiber is a myofibril. It is these chains of sarcomeres, or myofibrils, that are going to allow for contraction, since they are made up of cytoskeletal machinery. Therefore, our muscle fiber is going to need many of these myofibrils in order to be good at contraction.
First of all, there are hundreds of myofibrils in each muscle fiber. These are images of cross sections through muscle fibers... you'll see many dots on the cut edges; each of those dots is a myofibril.
Adjacent myofibrils line up evenly with each other. That means that the Z-lines of every sarcomere in one myofibril lines up with the Z-lines of every sarcomere in the adjacent myofibrils. Because of that, the I bands and the A bands in all the myofibrils within a muscle fiber are lined up. This is a difficult point to be able to understand with mental imagery. Take a look at the image here If you look up again at the drawing above of the sarcomere, you'll see that the sarcomere runs left and right, but the bands run up and down. Just like in this photo. This causes the muscle fibers to look striped, and this appearance is called striated. We can say that the muscle fibers are striated because they have striations (stripes).
Take a look at your textbook Figure 9.5 to see how adjacent myofibrils line up within a muscle fiber. For the sake of clarity, this figure only shows 9 myofibrils within the muscle fiber-- but there would be hundreds!
Now if you think back to the last web page, you'll remember that there are cell membrane invaginations called t-tubules. These t-tubules run into the muscle fiber at the Z-lines (although your book's Figure 9.5 doesn't really show it like that).
When the hypopolarizing stimulus of the spike in the T tubules is over, calcium ceases to be released by the cisternae of the sarcoplasmic reticulum and actively pumped into the longitudinal portion of the reticulum. The Ca pump that pumps Ca from the cytosol back into the sarcoplasmic reticulum is an ATPase that is phosporylated and dephosphorylated during the pumping process. It pumps two Ca ions for each ATP hydrolyzed. In muscle, the Ca ATPase accounts for nearly 90% of the membrane protein and therefore is capable of pumping Ca ions rapidly. Typically, the cytosolic Ca concentration is restored to resting levels within 30 milliseconds. When calcium is removed from the myofibrils, ATP replaces ADP on the myosin complex and the myosin-actin bond is broken. Because the muscle is elastic, it will be restored to its resting length in the absence of a further stimulus to release calcium. Shortening is an active process; lengthening is a passive process.
A single cycle of attachment, swivel, and detachment of the myosin head will produce a linear translation of the myofilaments of about 10 nm. If all cross-bridges in a myofibril cycle once synchronously, a relative movement equal to about 1% of the muscle length will occur, but obviously muscles shorten by more than 1%. The total shortening of a sarcomere during contraction may exceed 1,000 nm; therefore the relative movement of a thin and thick filament would be half this amount or 500 nm. To achieve this magnitude of change in total length when each cross-bridge cycle produces a 10-nm shortening, a minimum of 50 cycles must occur. The flexor muscles of the human upper arm can contract at the rate of 8 m/sec, during which they can shorten by as much as 10 cm. This contraction rate gives a contraction rate for the sarcomere of 160 nm/msec. If a stroke of the cross-bridge is taken to be 10 nm, then at this rate there will be a minimum of 16 strokes/msec. Thus, the swivel time for the cross-bridge must be of the order of 60 sec. Calculations for the frog's sartorius muscle, which can shorten at up to 4 cm/sec, indicate a swivel time of about 1 msec, but this contraction occurs at a lower temperature than those in mammals. In any case, it is clear that the swiveling of the cross-bridge must be a fast mechanical process. At the right is an animation that shows the repeated nature of the process.
The contractile characteristics and the mechanisms that cause contraction of vascular smooth muscle (VSM) are very different from cardiac muscle. VSM undergoes slow, sustained, tonic contractions, whereas cardiac muscle contractions are rapid and of relatively short duration (a few hundred milliseconds). While VSM contains actin and myosin, it does not have the regulatory protein troponin as is found in the heart.
Furthermore, the arrangement of actin and myosin in VSM is not organized into distinct bands as it is in cardiac muscle. This is not to imply that the contractile proteins of VSM are disorganized and not well-developed. They are actually highly organized and well-suited for their role in maintaining tonic contractions and reducing lumen diameter.
Contraction in VSM can be initiated by mechanical, electrical, and chemical stimuli. Passive stretching of VSM can cause contraction that originates from the smooth muscle itself and is therefore termed a myogenic response. Electrical depolarization of the VSM cell membrane also elicits contraction, most likely by opening voltage dependent calcium channels (L-type calcium channels), which causes an increase in the intracellular concentration of calcium. Finally, a number of chemical stimuli such as norepinephrine, angiotensin II, vasopressin, endothelin-1, and thromboxane A2 can cause contraction. Each of these substances bind to specific receptors on the VSM cell (or to receptors on the endothelium adjacent to the VSM), which then leads to VSM contraction.
The mechanism of contraction involves different signal transduction pathways, all of which converge to increase intracellular calcium.The mechanism by which an increase in intracellular calcium stimulates VSM contraction is illustrated in the figure to the right. An increase in free intracellular calcium can result from either increased flux of calcium into the cell through calcium channels or by release of calcium from internal stores (e.g., sarcoplasmic reticulum; SR). The free calcium binds to a special calcium binding protein called calmodulin. Calcium-calmodulin activates myosin light chain kinase (MLCK), an enzyme that is capable of phosphorylating myosin light chains (MLC) in the presence of ATP. Myosin light chains are 20-kD regulatory subunits found on the myosin heads. MLC phosphorylation leads to cross-bridge formation between the myosin heads and theactin filaments, and hence, smooth muscle contraction.
Intracellular calcium concentrations, therefore, are very important in regulating smooth muscle contraction. The concentration of intracellular calcium depends upon the balance between the calcium the enters the cells, the calcium that is released by intracellular storage sites (e.g., SR), and removal of calcium either back into storage sites or out of the cell. Calcium is re-sequestered by the SR by a ATP-dependent calcium pump. Calcium is removed from the cell to the external environment by either a ATP-dependent calcium pump or by the sodium-calcium exchanger.
ATP is the immediate source of energy for muscle contraction. Although a muscle fiber contains only enough ATP to power a few twitches, its ATP "pool" is replenished as needed. There are three sources of high-energy phosphate to keep the ATP pool filled.
The phosphate group in creatine phosphate is attached by a "high-energy" bond like that in ATP. Creatine phosphate derives its high-energy phosphate from ATP and can donate it back to ADP to form ATP.
Skeletal muscle fibers contain about 1% glycogen. The muscle fiber can degrade this glycogen by glycogenolysis producing glucose-1-phosphate. This enters the glycolytic pathway to yield two molecules of ATP for each pair of lactic acid molecules produced. Not much, but enough to keep the muscle functioning if it fails to receive sufficient oxygen to meet its ATP needs by respiration.
Cellular respiration not only is required to meet the ATP needs of a muscle engaged in prolonged activity (thus causing more rapid and deeper breathing), but is also required afterwards to enable the body to resynthesize glycogen from the lactic acid produced earlier (deep breathing continues for a time after exercise is stopped). The body must repay its oxygen debt.
The properties of both red and white muscles are summarized in Table. The properties of slow muscle fibers make them most suited to extended periods of contraction where a minimum force is required, e.g., in maintenance of posture. Fast muscle fibers are better suited to short periods of rapid contraction at higher forces, e.g., in sprint running. In fact, during exercise training there may be a differential effect on the two types of muscles. Strength training leads to hypertrophy of mainly white muscles with conversion of FOG to FG fibers. The number of fibers does not increase, but the size of fibers and the number of myofibrils do increase. This increases both the strength and velocity of contraction. Endurance training apparently affects mainly red muscle fibers, causing an increase in concentration of the enzymes of oxidative phosphorylation, an increase in the vascularization of the muscle and conversion of FG to FOG fibers, but no change in the ratio of fast to slow fibers and no change in muscle size.
· The action potential that triggers the heartbeat is generated within the heart itself. Motor nerves (of the autonomic nervous system) do run to the heart, but their effect is simply to modulate — increase or decrease — the intrinsic rate and the strength of the heartbeat. Even if the nerves are destroyed (as they are in a transplanted heart), the heart continues to beat.
· Significance: All the fibers contract in a synchronous wave that sweeps from the atria down through the ventricles and pumps blood out of the heart. Anything that interferes with this synchronous wave (such as damage to part of the heart muscle from a heart attack) may cause the fibers of the heart to beat at random — called fibrillation. Fibrillation is the ultimate cause of most deaths and its reversal is the function of defibrillators that are part of the equipment in ambulances, hospital emergency rooms, and — recently — even on U.S. air lines.
Below: the human heart, with a schematic view of the pathway of blood through the lungs and internal organs. Oxygenated blood is shown in red; deoxygenated blood in blue. Note that the blood draining the stomach, spleen, and intestines passes through the liver before it is returned to the heart. Here surplus or harmful materials picked up from those organs can be removed before the blood returns to the general circulation.
Structurally, cardiac muscle is similar to skeletal muscle in that it is striated, having both thick and thin filaments. It has a well-developed T tubule system, although the sarcoplasmic reticulum is not as large or as extensive as in skeletal muscle. Unlike those in skeletal muscle, the triads of cardiac muscle of humans are located at the Z line, giving only one per sarcomere. The mechanism of excitation-contraction coupling is the same as for skeletal muscle: The membrane action potential leads to an increase in Ca++ around the myofilaments that activates myosin-ATPase and leads to sliding of the thin and thick filaments. The source of the calcium is different in cardiac muscle. Because the sarcoplasmic reticulum is poorly developed, it cannot sequester the large amount of calcium that skeletal muscle can. Therefore, much of the calcium for contraction must come from extracellular sources; it comes in during the action potential.
There are a large number of different kinds of cells in cardiac muscle. These include cells of the sinoatrial node, the atrioventricular node, the atrium, the bundle of His, and the ventricle, each with a differently shaped action potential. The details of these differences are beyond the scope of this treatment. For our purposes, it is convenient to distinguish two kinds of cardiac muscle cells: pacemaker cells, like the Purkinje fibers, and contractile cells. Examples of a Purkinje fiber action potential (A) and a contractile cell action potential (B) Both action potentials are much longer in duration than spikes in nerve cells and skeletal muscle cells, 0.5 sec compared to 0.5 to 5.0 msec. The hypopolarizing phase of the Purkinje fiber's action potential is not different from that in skeletal muscle, and it appears to have the same ionic mechanism, i.e., a dramatic increase in sodium conductance. The contractile cell's action potential has two rising phases, a rapidly rising phase, like that in the Purkinje fiber, and a more slowly rising phase. The fast phase has the same mechanism as the rising phase of Purkinje fiber action potentials, but the slower phase is the result of a slow inward current, carried mostly by Ca++. Calcium current activation occurs at a more hypopolarized level of the membrane potential than does sodium activation, and the inactivation of the calcium current is less rapid by about two orders of magnitude.
The long plateau of the action potential in cardiac muscle serves two functions: It provides a more prolonged contraction without resorting to tetanus, and it provides a longer refractory period to prevent the heart from contracting prematurely. This plateau is produced by a number of factors, the most important of which is a decrease in potassium conductance with hypopolarization, followed by a slowly developing increase that brings the potassium conductance to a final value just slightly greater than resting levels in Purkinje fibers and to resting levels in contractile cells in about 300 msec. A change in membrane conductance with changes in membrane potential is called rectification by biophysicists. This change in potassium conductance is called anomalous rectification.
Cardiac muscle behaves much like skeletal muscle, but it exerts a passive tension when stretched at much shorter lengths. In fact, when the muscle is stretched from a length even shorter than resting length, there is a resistance to the stretch. In other words, cardiac muscle experiences elastic tension even at resting length (skeletal muscle does not). In addition, the maximum developed tension in cardiac muscle occurs, not at the resting length, but when it is stretched beyond resting length. The result is that when more blood returns to the heart from the veins, the muscle fibers of the heart will be stretched more, and the blood will automatically be pumped out more forcefully than when the heart is just normally full. This is the basis of the Frank-Starling mechanism in the heart.
It is used to find out whether your kidneys are working normally. A combination of blood and urine creatinine levels may be used to calculate a "creatinine clearance". This measures how effectively your kidneys are filtering small molecules like creatinine out of your blood.
Urine creatinine may also be used with a variety of other urine tests as a correction factor. Since it is produced and removed at a relatively constant rate, the amount of creatinine in urine can be compared to the amount of another substance being measured. Examples of this are when creatinine is measured with protein to calculate a urine protein/creatinine ratio (UP/CR) and when it is measured with microalbumin to calculate microalbumin/creatinine ratio (also known as albumin/creatinine ratio, ACR). These tests are used to evaluate kidney function as well as to detect other urinary tract disorders.
Serum creatinine measurements along with age, weight, and gender are used to calculate the estimated glomerular filtration rate (eGFR), which is used as a screening test to look for evidence of kidney damage.
Low levels of creatinine are not common and are not usually a cause for concern. As creatinine levels are related to the amount of muscle the person has, low levels may be a consequence of decreased muscle mass (such as in the elderly), but may also be occasionally found in advanced liver disease.
Random urine creatinine levels have no standard reference ranges. They are usually used with other tests to reference levels of other substances measured in the urine. Some examples include the microalbuminuria test and urine protein test.
In general, creatinine levels will stay the same if you eat a normal diet. However, eating large amounts of meat may cause short-lived increases in blood creatinine levels. Taking creatine supplements may also increase creatinine.
There are a few drugs that interfere with the creatinine test, although there are some drugs that can cause some impairment in kidney function. Your creatinine levels may be monitored if you are taking one of these drugs.
CK is often determined routinely in a medical laboratory. It is also determined specifically in patients with chest pain or if acute renal failure is suspected. Normal values are usually between 60 and 400 IU/L, where one unit is enzyme activity, more specifically the amount of enzyme that will catalyze 1 μmol of substrate per minute under specified conditions (temperature, pH, substrate concentrations and activators. This test is not specific for the type of CK that is elevated.
Elevation of CK is an indication of damage to muscle. It is therefore indicative of injury, rhabdomyolysis, myocardial infarction, myositis and myocarditis. The use of statin medications, which are commonly used to decrease serum cholesterol levels, may be associated with elevation of the CPK level in about 1% of the patients taking these medications, and with actual muscle damage in a much smaller proportion.
There is an inverse relationship in the serum levels of T3 and CK in thyroid disease. In hypothyroid patients, with decrease in serum T3 there is a significant increase in CK. Therefore, the estimation of serum CK is considered valuable in screening for hypothyroid patients.
Isoenzyme determination has been used extensively as an indication for myocardial damage in heart attacks. Troponin measurement has largely replaced this in many hospitals, although some centers still rely on CK-MB.
Connective tissue (CT) is a kind of biological tissue that supports, connects, or separates different types of tissues and organs of the body. It is one of the four general classes of biological tissues—the others of which are epithelial, muscular, and nervous tissues.
All CT has three main components: cells, fibers, and extracellular matrices, all immersed in the body fluids.
Connective tissue can be broadly subdivided into connective tissue proper, special connective tissue, and series of other, less classifiable types of connective tissues. Connective tissue proper consists of loose connective tissue and dense connective tissue (which is further subdivided into dense regular and dense irregular connective tissues.) Special connective tissue consists of reticular connective tissue, adipose tissue, cartilage, bone, and blood. Other kinds of connective tissues include fibrous, elastic, and lymphoid connective tissues.
Fibroblasts are the cells responsible for the production of some CT.
Type-I collagen, is present in many forms of connective tissue, and makes up about 25% of the total protein content of the mammalian body.
Most mammalian cells are located in tissues where they are surrounded by a complex extracellular matrix (ECM) often referred to as “connective tissue.” The ECM contains three major classes of biomolecules:
(1)the structural proteins, collagen, elastin, and fibrillin;
(2) certain specialized proteins such as fibrillin, fibronectin, and laminin; and
(3) proteoglycans, whose chemical natures are described below.
The extracellular matrix
The ECM has been found to be involved in many normal and pathologic processes—eg, it plays important roles in development, in inflammatory states, and in the spread of cancer cells. Involvement of certain components of the ECM has been documented in both rheumatoid arthritis and osteoarthritis. Several diseases (eg, osteogenesis imperfecta and a number of types of the Ehlers-Danlos syndrome) are due to genetic disturbances of the synthesis of collagen. Specific components of proteoglycans (the glycosaminoglycans; GAGs) are affected in the group of genetic disorders known as the mucopolysaccharidoses.
Changes occur in the ECM during the aging process. This chapter describes the basic biochemistry of the three major classes of biomolecules found in the ECM and illustrates their biomedical significance.
Major biochemical features of two specialized forms of ECM—bone and cartilage—and of a number of diseases involving them are also briefly considered.
Functions of connective tissue
- Storage of energy
- Protection of organs
- Provision of structural framework for the body
- Connection of body tissues
- Connection of epithelial tissues to muscle tissues
Characteristics of connective tissue and fiber types
Cells are spread through an extracellular fluid.
Ground substance - A clear, colorless, and viscous fluid containing glycosaminoglycans and proteoglycans to fix the bodywater and the collagen fibers in the intercellular spaces. Ground substance slows the spread of pathogens.
Fibers. Not all types of CT are fibrous. Examples include adipose tissue and blood. Adipose tissue gives "mechanical cushioning" to our body, among other functions. Although there is no dense collagen network in adipose tissue, groups of adipose cells are kept together by collagen fibers and collagen sheets in order to keep fat tissue under compression in place (for example, the sole of the foot). The matrix of blood is plasma.
Both the ground substance and proteins (fibers) create the matrix for CT.
Types of fibers:Tissue → Purpose → Components → Location
Collagenous fibers → Alpha polypeptide chains → tendon, ligament, skin, cornea, cartilage, bone, blood vessels, gut, and intervertebral disc.
Elastic fibers → elastic microfibril & elastin → extracellular matrix
Reticular fibers → Type-III collagen → liver, bone marrow, lymphatic organs.
Structure and functions of collagen.
It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content. Collagen, in the form of elongated fibrils, is mostly found in fibrous tissues such as tendon, ligament and skin, and is also abundant in cornea, cartilage, bone, blood vessels, the gut, and intervertebral disc. The fibroblast is the most common cell which creates collagen. In muscle tissue, it serves as a major component of the endomysium.
Fig.Functions of collagen
Collagen constitutes one to two percent of muscle tissue, and accounts for 6% of the weight of strong, tendinous muscles. Gelatin, which is used in food and industry, is collagen that has been irreversibly hydrolyzed. Collagen is composed of a triple helix, which generally consists of two identical chains (α1) and an additional chain that differs slightly in its chemical composition (α2). The amino acid composition of collagen is atypical for proteins, particularly with respect to its high hydroxyproline content. The most common motifs in the amino acid sequence of collagen are glycine-proline-X and glycine-X-hydroxyproline, where X is any amino acid other than glycine, proline or hydroxyproline.
Figure 1. Collagen structures
First, a three dimensional stranded structure is assembled, with the amino acids glycine and proline as its principal components. This is not yet collagen but its precursor, procollagen. A recent study shows that vitamin C must have an important role in its synthesis. Prolonged exposure of cultures of human connective-tissue cells to ascorbate induced an eight-fold increase in the synthesis of collagen with no increase in the rate of synthesis of other proteins. Since the production of procollagen must precede the production of collagen, vitamin C must have a role in this step. The conversion involves a reaction that substitutes a hydroxyl group, OH, for a hydrogen atom, H, in the proline residues at certain points in the polypeptide chains, converting those residues to hydroxyproline. This hydroxylation reaction organizes the chains in the conformation necessary for them to form a triple helix. The hydroxylation, next, of the residues of the amino acid lysine, transforming them to hydroxylysine, is then needed to permit the cross-linking of the triple helices into the fibers and networks of the tissues.
These hydroxylation reactions are catalyzed by two different enzymes: prolyl-4-hydroxylase and lysyl-hydroxylase. Vitamin C also serves with them in inducing these reactions. in this service, one molecule of vitamin C is destroyed for each H replaced by OH. The synthesis of collagen occurs inside and outside of the cell. The formation of collagen which results in fibrillary collagen (most common form) is discussed here. Meshwork collagen, which is often involved in the formation of filtration systems is the other form of collagen. It should be noted that all types of collagens are triple helixes, and the differences lie in the make-up of the alpha peptides created in step 2.
1. Transcription of mRNA: There are approximately 34 genes associated with collagen formation, each coding for a specific mRNA sequence, and typically have the "COL" prefix. The beginning of collagen synthesis begins with turning on genes which are associated with the formation of a particular alpha peptide (typically alpha 1, 2 or 3).
2. Pre-pro-peptide Formation: Once the final mRNA exits from the cell nucleus and enters into the cytoplasm it links with the ribosomal subunits and the process of translation occurs. The early/first part of the new peptide is known as the signal sequence. The signal sequence on the N-terminal of the peptide is recognized by a signal recognition particle on the endoplasmic reticulum, which will be responsible for directing the pre-pro-peptide into the endoplasmic reticulum. Therefore, once the synthesis of new peptide is finished, it goes directly into the endoplasmic reticulum for post-translational processing. Note that it is now known as pre-pro-collagen.
Fig. Biosynthesis of collagen from Preprocollagen
3. Alpha Peptide to Procollagen: Three modifications of the pre-pro-peptide occur leading to the formation of the alpha peptide. Secondly, the triple helix known as procollagen is formed before being transported in a transport vesicle to the golgi apparatus. 1) The signal peptide on the N-terminal is dissolved, and the molecule is now known as propeptide (not procollagen). 2) Hydroxylation of lysines and prolines on propeptide by the enzymes prolyl hydroxylase and lysyl hydroxylase (to produce hydroxyproline and hydroxylysine) occurs to aid crosslinking of the alpha peptides. It is this enzymatic step that requires vitamin C as a cofactor. In scurvy, the lack of hydroxylation of prolines and lysines causes a looser triple helix (which is formed by 3 alpha peptides). 3) Glycosylation occurs by adding either glucose or galactose monomers onto the hydroxy groups that were placed onto lysines, but not on prolines. From here the hydroxylated and glycosylated propeptide twists towards the left very tightly and then three propeptides will form a triple helix. It is important to remember that this molecule, now known as procollagen (not propeptide) is composed of a twisted portion (center) and two loose ends on either end. At this point the procollagen is packaged into a transfer vesicle destined for the golgi apparatus.
4. Golgi Apparatus Modification: In the golgi apparatus, the procollagen goes through one last post-translational modification before being secreted out of the cell. In this step oligosaccharides (not monosaccharides like in step 3) are added, and then the procollagen is packaged into a secretory vesicle destined for the extracellular space.
5. Formation of Tropocollagen: Once outside the cell, membrane bound enzymes known as collagen peptidases, remove the "loose ends" of the procollagen molecule. What is left is known as tropocollagen. Defect in this step produces one of the many collagenopathies known as Ehlers-Danlos syndrome. This step is absent when synthesizing type III, a type of fibrilar collagen.
6. Formation of the Collagen Fibril: Lysyl oxidase an extracellular enzyme produces the final step in the collagen synthesis pathway. This enzyme acts on lysines and hydroxylysines producing aldehyde groups, which will eventually undergo covalent bonding between tropocollagen molecules. This polymer of tropocollogen is known as a collagen fibril.
Collagen has an unusual amino acid composition and sequence:
· Proline (Pro) makes up about 17% of collagen
· Collagen contains two uncommon derivative amino acids not directly inserted during translation. These amino acids are found at specific locations relative to glycine and are modified post-translationally by different enzymes, both of which require vitamin C as a cofactor.
o Hydroxyproline (Hyp), derived from proline.
Most collagen forms in a similar manner, but the following process is typical for type I:
1. Inside the cell
1. Two types of peptide chains are formed during translation on ribosomes along the rough endoplasmic reticulum (RER): alpha-1 and alpha-2 chains. These peptide chains (known as preprocollagen) have registration peptides on each end and a signal peptide.
2. Polypeptide chains are released into the lumen of the RER.
3. Signal peptides are cleaved inside the RER and the chains are now known as pro-alpha chains.
5. Glycosylation of specific hydroxylysine residues occurs.
6. Triple ɣ helical structure is formed inside the endoplasmic reticulum from each two alpha-1 chains and one alpha-2 chain.
2. Outside the cell
1. Registration peptides are cleaved and tropocollagen is formed by procollagen peptidase.
2. Multiple tropocollagen molecules form collagen fibrils, via covalent cross-linking (aldol reaction) by lysyl oxidase which links hydroxylysine and lysine residues. Multiple collagen fibrils form into collagen fibers.
Vitamin C deficiency causes scurvy, a serious and painful disease in which defective collagen prevents the formation of strong connective tissue. Gums deteriorate and bleed, with loss of teeth; skin discolors, and wounds do not heal. Prior to the eighteenth century, this condition was notorious among long duration military, particularly naval, expeditions during which participants were deprived of foods containing Vitamin C.
Many bacteria and viruses have virulence factors which destroy collagen (such as the enzyme collagenase) or interfere with its production.
The tropocollagen or collagen molecule is a subunit of larger collagen aggregates such as fibrils. At approximately 300 nm long and 1.5 nm in diameter, it is made up of three polypeptide strands (called alpha peptides, see step 2), each possessing the conformation of a left-handed helix (its name is not to be confused with the commonly occurring alpha helix, a right-handed structure). These three left-handed helices are twisted together into a right-handed coiled coil, a triple helix or "super helix", a cooperative quaternary structure stabilized by numerous hydrogen bonds. With type I collagen and possibly all fibrillar collagens if not all collagens, each triple-helix associates into a right-handed super-super-coil referred to as the collagen microfibril. Each microfibril is interdigitated with its neighboring microfibrils to a degree that might suggest they are individually unstable, although within collagen fibrils, they are so well ordered as to be crystalline.
A distinctive feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits. The sequence often follows the pattern Gly-Pro-X or Gly-X-Hyp, where X may be any of various other amino acid residues. Proline or hydroxyproline constitute about 1/6 of the total sequence. With glycine accounting for the 1/3 of the sequence, this means approximately half of the collagen sequence is not glycine, proline or hydroxyproline, a fact often missed due to the distraction of the unusual GX1X2 character of collagen alpha-peptides. The high glycine content of collagen is important with respect to stabilization of the collagen helix as this allows the very close association of the collagen fibers within the molecule, facilitating hydrogen bonding and the formation of intermolecular cross-links. This kind of regular repetition and high glycine content is found in only a few other fibrous proteins, such as silk fibroin. About 75-80% of silk is (approximately) -Gly-Ala-Gly-Ala- with 10% serine, and elastin is rich in glycine, proline, and alanine (Ala), whose side group is a small methyl group. Such high glycine and regular repetitions are never found in globular proteins save for very short sections of their sequence. Chemically reactive side groups are not needed in structural proteins, as they are in enzymes and transport proteins; however, collagen is not quite just a structural protein. Due to its key role in the determination of cell phenotype, cell adhesion, tissue regulation and infrastructure, many sections of its nonproline-rich regions have cell or matrix association / regulation roles. The relatively high content of proline and hydroxyproline rings, with their geometrically constrained carboxyl and (secondary) amino groups, along with the rich abundance of glycine, accounts for the tendency of the individual polypeptide strands to form left-handed helices spontaneously, without any intrachain hydrogen bonding.
Because glycine is the smallest amino acid with no side chain, it plays a unique role in fibrous structural proteins. In collagen, Gly is required at every third position because the assembly of the triple helix puts this residue at the interior (axis) of the helix, where there is no space for a larger side group than glycine’s single hydrogen atom. For the same reason, the rings of the Pro and Hyp must point outward. These two amino acids help stabilize the triple helix—Hyp even more so than Pro; a lower concentration of them is required in animals such as fish, whose body temperatures are lower than most warm-blooded animals. Lower proline and hydroxyproline contents are characteristic of cold-water, but not warm-water fish; the latter tend to have similar proline and hydroxyproline contents to mammals. The lower proline and hydroxproline contents of cold-water fish and other poikilotherm animals leads to their collagen having a lower thermal stability than mammalian collagen. This lower thermal stability means that gelatin derived from fish collagen is not suitable for many food and industrial applications.
The tropocollagen subunits spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. In the fibrillar collagens, the molecules are staggered from each other by about 67 nm (a unit that is referred to as ‘D’ and changes depending upon the hydration state of the aggregate). Each D-period contains four plus a fraction collagen molecules, because 300 nm divided by 67 nm does not give an integer (the length of the collagen molecule divided by the stagger distance D). Therefore, in each D-period repeat of the microfibril, there is a part containing five molecules in cross-section, called the “overlap”, and a part containing only four molecules, called the "gap". The triple-helices are also arranged in a hexagonal or quasihexagonal array in cross-section, in both the gap and overlap regions.
There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices forming well organized aggregates (such as fibrils). Larger fibrillar bundles are formed with the aid of several different classes of proteins (including different collagen types), glycoproteins and proteoglycans to form the different types of mature tissues from alternate combinations of the same key players. Collagen's insolubility was a barrier to the study of monomeric collagen until it was found that tropocollagen from young animals can be extracted because it is not yet fully crosslinked. However, advances in microscopy techniques (i.e. electron microscopy (EM) and atomic force microscopy (AFM)) and X-ray diffraction have enabled researchers to obtain increasingly detailed images of collagen structure in situ. These later advances are particularly important to better understanding the way in which collagen structure affects cell-cell and cell-matrix communication, and how tissues are constructed in growth and repair, and changed in development and disease. For example using AFM –based nanoindentation it has been shown that a single collagen fibril is a heterogeneous material along its axial direction with significantly different mechanical properties in its gap and overlap regions, correlating with its different molecular organizations in these two regions.
Collagen fibrils/aggregates are arranged in different combinations and concentrations in various tissues to provide varying tissue properties. In bone, entire collagen triple helices lie in a parallel, staggered array. 40 nm gaps between the ends of the tropocollagen subunits (approximately equal to the gap region) probably serve as nucleation sites for the deposition of long, hard, fine crystals of the mineral component, which is (approximately) Ca10(OH)2(PO4)6. Type I collagen gives bone its tensile strength.
Collagen occurs in many places throughout the body. Over 90% of the collagen in the body, however, is of type I.
So far, 28 types of collagen have been identified and described. The five most common types are:
· Collagen I: skin, tendon, vascular ligature, organs, bone (main component of the organic part of bone)
· Collagen II: cartilage (main component of cartilage)
· Collagen III: reticulate (main component of reticular fibers), commonly found alongside type I.
· Collagen IV: forms bases of cell basement membrane
· Collagen V: cell surfaces, hair and placenta
Collagen-related diseases most commonly arise from genetic defects or nutritional deficiencies that affect the biosynthesis, assembly, postranslational modification, secretion, or other processes involved in normal collagen production.
Tabl. Types Of Collagen
A Number of Genetic Diseases Result From Abnormalities in the Synthesis of Collagen
About 30 genes encode collagen, and its pathway of biosynthesis is complex, involving at least eight enzyme-catalyzed posttranslational steps. Thus, it is not surprising that a number of diseases are due to mutations in collagen genes or in genes encoding some of the enzymes involved in these posttranslational
modifications. The diseases affecting bone (eg, osteogenesis imperfecta) and cartilage (eg, the chondrodysplasias) will be discussed later in this chapter.
Ehlers-Danlos syndrome comprises a group of inherited disorders whose principal clinical features are hyperextensibility of the skin, abnormal tissue fragility,
and increased joint mobility. The clinical picture is variable, reflecting underlying extensive genetic heterogeneity.
At least 10 types have been recognized, most but not all of which reflect a variety of lesions in the synthesis of collagen. Type IV is the most serious because of its tendency for spontaneous rupture of arteries or the bowel, reflecting abnormalities in type III collagen.
Patients with type VI, due to a deficiency of lysyl hydroxylase, exhibit marked joint hypermobility and a tendency to ocular rupture. A deficiency of procollagen
N-proteinase, causing formation of abnormal thin, irregular collagen fibrils, results in type VIIC, manifested by marked joint hypermobility and soft skin.
Alport syndrome is the designation applied to a number of genetic disorders (both X-linked and autosomal) affecting the structure of type IV collagen fibers, the major collagen found in the basement membranes of the renal glomeruli (see discussion of laminin, below). Mutations in several genes encoding type IV collagen fibers have been demonstrated. The presenting sign is hematuria, and patients may eventually develop end-stage renal disease. Electron microscopy reveals characteristic abnormalities of the structure of the basement membrane and lamina densa.
In epidermolysis bullosa, the skin breaks and blisters as a result of minor trauma. The dystrophic form is due to mutations in COL7A1, affecting the structure of type VII collagen. This collagen forms delicate fibrils that anchor the basal lamina to collagen fibrils in the dermis. These anchoring fibrils have been shown to be markedly reduced in this form of the disease, probably resulting in the blistering. Epidermolysis bullosa simplex, another variant, is due to mutations in keratin 5.
Scurvy affects the structure of collagen. However, it is due to a deficiency of ascorbic acid and is not a genetic disease. Its major signs are bleeding gums, subcutaneous hemorrhages, and poor wound healing. These signs reflect impaired synthesis of collagen due to deficiencies of prolyl and lysyl hydroxylases, both of which require ascorbic acid as a cofactor.
Osteoporosis - Not inherited genetically, brought on with age, associated with reduced levels of collagen in the skin and bones, growth hormone injections are being researched as a possible treatment to counteract any loss of collagen.
Knobloch syndrome - Caused by a mutation in the collagen XVIII gene, patients present with protrusion of the brain tissue and degeneration of the retina, an individual who has family members suffering from the disorder are at an increased risk of developing it themselves as there is a hereditary link.
Collagen is one of the long, fibrous structural proteins whose functions are quite different from those of globular proteins such as enzymes. Tough bundles of collagen called collagen fibers are a major component of the extracellular matrix that supports most tissues and gives cells structure from the outside, but collagen is also found inside certain cells. Collagen has great tensile strength, and is the main component of fascia, cartilage, ligaments, tendons, bone and skin. Along with soft keratin, it is responsible for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging. It strengthens blood vessels and plays a role in tissue development. It is present in the cornea and lens of the eye in crystalline form.
If collagen is sufficiently denatured, e.g. by heating, the three tropocollagen strands separate partially or completely into globular domains, containing a different secondary structure to the normal collagen polyproline II (PPII), e.g. random coils. This process describes the formation of gelatin, which is used in many foods, including flavored gelatin desserts. Besides food, gelatin has been used in pharmaceutical, cosmetic, and photography industries. From a nutritional point of view, collagen and gelatin are a poor-quality sole source of protein since they do not contain all the essential amino acids in the proportions that the human body requires—they are not 'complete proteins' (as defined by food science, not that they are partially structured). Manufacturers of collagen-based dietary supplements claim that their products can improve skin and fingernail quality as well as joint health. However, mainstream scientific research has not shown strong evidence to support these claims. Individuals with problems in these areas are more likely to be suffering from some other underlying condition (such as normal aging, dry skin, arthritis etc.) rather than just a protein deficiency.
From the Greek for glue, kolla, the word collagen means "glue producer" and refers to the early process of boiling the skin and sinews of horses and other animals to obtain glue. Collagen adhesive was used by Egyptians about 4,000 years ago, and Native Americans used it in bows about 1,500 years ago. The oldest glue in the world, carbon-dated as more than 8,000 years old, was found to be collagen—used as a protective lining on rope baskets and embroidered fabrics, and to hold utensils together; also in crisscross decorations on human skulls. Collagen normally converts to gelatin, but survived due to the dry conditions. Animal glues are thermoplastic, softening again upon reheating, and so they are still used in making musical instruments such as fine violins and guitars, which may have to be reopened for repairs—an application incompatible with tough, synthetic plastic adhesives, which are permanent. Animal sinews and skins, including leather, have been used to make useful articles for millennia.
Elastin – main protein of elastic fibrils, structure and biological role.
Elastin is a protein in connective tissue that is elastic and allows many tissues in the body to resume their shape after stretching or contracting. Elastin helps skin to return to its original position when it is poked or pinched. Elastin is also an important load-bearing tissue in the bodies of vertebrates and used in places where mechanical energy is required to be stored. In humans, elastin is encoded by the ELN gene.
This gene encodes a protein that is one of the two components of elastic fibers. The encoded protein is rich in hydrophobic amino acids such as glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues. Multiple transcript variants encoding different isoforms have been found for this gene. The other name for elastin is tropoelastin. The characterization of disorder is consistent with an entropy-driven mechanism of elastic recoil. It is concluded that conformational disorder is a constitutive feature of elastin structure and function.
Deletions and mutations in this gene are associated with supravalvular aortic stenosis (SVAS) and autosomal dominant cutis laxa. Other associated defects in elastin include Marfan's Syndrome and emphysema caused by α1-antitrypsin deficiency.
Elastic fiber is composed of the protein fibrillin and elastin made of simple amino acids such as glycine, valine, alanine, and proline. The total elastin ranges from 58 to 75% of the weight of the dry defatted artery in normal canine arteries. Comparison between fresh and digested tissues shows that, at 35% strain, a minimum of 48% of the arterial load is carried by elastin, and a minimum of 43% of the change in stiffness of arterial tissue is due to the change in elastin stiffness. Elastin is made by linking many soluble tropoelastin protein molecules, in a reaction catalyzed by lysyl oxidase, to make a massive insoluble, durable cross-linked array. The amino acid responsible for these cross-links is lysine. Tropoelastin is a specialized protein with a molecular weight of 64 to 66 kDa, and an irregular or random coil conformation made up of 830 amino acids.
Elastin serves an important function in arteries as a medium for pressure wave propagation to help blood flow and is particularly abundant in large elastic blood vessels such as the aorta. Elastin is also very important in the lungs, elastic ligaments, the skin, and the bladder, elastic cartilage. It is present in all vertebrates above the jawless fish.
Table 1 summarizes the main differences between collagen and elastin.
Table 1. Major differences between collagen and elastin.
In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue protein with rubber-like properties. Elastic fibers composed of elastin and glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments.
They can be stretched to several times their normal length, but recoil to their original shape when the stretching force is relaxed.
A. Structure of elastin
Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin, which is a linear polypeptide composed of about 700 amino acids that are primarily small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich in proline and lysine, but contains only a little hydroxyproline and hydroxy lysine.
Tropoelastin is secreted by the cell into the extracellular space. There it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the tropoelastin poly peptides are oxidatively deaminated by lysyl oxidase, forming allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain from the same or neighboring polypeptides form a desmosine cross-link (Figure 4.12). This produces elastin—an extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Figure 4.13). Mutations in the fibrillin-1 protein are responsible for Marfan syndrome—a connective tissue disorder characterized by impaired structural integrity in the skeleton, the eye, and the cardiovascular system. With this disease, abnormal fibrillin protein is incorporated into microfibrils along with normal fibrillin, inhibiting the formation of functional microfibrils. [Note: Patients with OI, EDS, or Marfan syndrome may have blue sclera due to tissue thinning that allows underlying pigment to show through.]
B. Role of 1-antitrypsin in elastin degradation
1. 1-Antitrypsin: Blood and other body fluids contain a protein, α1-antitrypsin (α1-AT, A1AT, currently also called α1-antiproteinase), that inhibits a number of proteolytic enzymes (also called proteases or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was originally named α1-antitrypsin because it inhibits the activity of trypsin (a proteolytic enzyme synthesized as trypsinogen by the pancreas] α1-AT comprises more than 90% of the α1-globulin fraction of normal plasma. α1-AT has the important physiologic role of inhibiting neutrophil elastase––a powerful protease that is released into the extracellular space, and degrades elastin of alveolar walls, as well as other structural proteins in a variety of tissues (Figure 4.14). Most of the α1-AT found in plasma is synthesized and secreted by the liver. The remainder is synthesized by several tissues, including monocytes and alveolar macrophages, which may be important in the prevention of local tissue injury by elastase.
2. Role of 1-AT in the lungs: In the normal lung, the alveoli are chronically exposed to low levels of neutrophil elastase released from activated and degenerating neutrophils. This proteolytic activity can destroy the elastin in alveolar walls if unopposed by the action of α1-AT, the most important inhibitor of neutrophil elastase (see Figure 4.14). Because lung tissue cannot regenerate, emphysema results from the destruction of the connective tissue of alveolar walls.
3. Emphysema resulting from 1-AT deficiency:
In the United States, approximately 2–5% of patients with emphysema are predisposed to the disease by inherited defects in α1-AT. A number of different mutations in the gene for α1-AT are known to cause a deficiency of this protein, but one single purine base mutation (GAG → AAG, resulting in the substitution of lysine for glutamic acid at position 342 of the protein) is clinically the most widespread.
The polymerization of the mutated protein in the endoplasmic reticulum of hepatocytes causes decreased secretion of
α1-AT by the liver. The accumulated polymer may result in cirrhosis (scarring of the liver). In the United States, the α1-AT mutation is most common in Caucasians of Northern European ancestry.
An individual must inherit two abnormal α1-AT alleles to be at risk for the development of emphysema. In a heterozygote, with one normal and one defective gene, the levels of α1-AT are sufficient to protect the alveoli from damage. [Note: A specific α1-AT methionine is required for the binding of the inhibitor to its target proteases.
Smoking causes the oxidation and subsequent inactivation of that methionine residue, thereby rendering the inhibitor powerless to neutralize elastase. Smokers with α1-AT deficiency, therefore, have a considerably elevated rate of lung destruction and a poorer survival rate than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be reversed by augmentation therapy—weekly intravenous administration of α1-AT. The α1-AT diffuses from the blood into the lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial cells.
§ cross-shaped glycoprotein
§ 3 polypeptides a, b1, b2
§ carbohydrate (13% by weight)
§ Mr 900K
§ separate binding domains
§ collagen IV
§ heparin sulphate
§ cell binding
§ cell specific binding - liver, nerve
§ cell surface receptor
§ cell adhesion
§ migration pathways
§ stimulates growth of axons
§ development and regeneration
§ basal laminae
§ most abundant linking glycoprotein
PROTEOGLYCANS & GLYCOSAMINOGLYCANS
The Glycosaminoglycans Found in Proteoglycans Are Built Up of Repeating Disaccharides
Proteoglycans are proteins that contain covalently linked glycosaminoglycans. A number of them have been characterized and given names such as syndecan, betaglycan, serglycin, perlecan, aggrecan, versican, decorin, biglycan, and fibromodulin. They vary in tissue distribution, nature of the core protein, attached glycosaminoglycans, and function. The proteins bound covalently to glycosaminoglycans are called “core proteins”; they have proved difficult to isolate and characterize, but the use of recombinant DNA technology is beginning to yield important information about their structures. The amount of carbohydrate in a proteoglycan is usually much greater than is found in a glycoprotein and may comprise up to 95% of its weight. Figures 2 and 3 show the general structure of one particular proteoglycan, aggrecan, the major type found in cartilage.
Figure 2. Dark field electron micrograph of a proteoglycan aggregate in which the proteoglycan subunits and filamentous backbone are particularly well extended.
It is very large (about 2 × 103 kDa), with its overall structure resembling that of a bottle brush. It contains a long strand of hyaluronic acid (one type of GAG) to which link proteins are attached noncovalently.
Figure 3. Schematic representation of the proteoglycan aggrecan.
In turn, these latter interact noncovalently with core protein molecules from which chains of other GAGs (keratan sulfate and chondroitin sulfate in this case) project. More details on this macromolecule are given when cartilage is discussed below.
There are at least seven glycosaminoglycans (GAGs): hyaluronic acid, chondroitin sulfate, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate. A GAG is an unbranched polysaccharide made up of repeating disaccharides, one component of which is always an amino sugar (hence the name GAG), either D-glucosamine or D-galactosamine. The other component of the repeating disaccharide (except in the case of keratan sulfate) is a uronic acid, either L-glucuronic acid (GlcUA) or its 5′-epimer, L-iduronic acid (IdUA). With the exception of hyaluronic acid, all the GAGs contain sulfate groups, either as O-esters or as N-sulfate (in heparin and heparan sulfate).
Hyaluronic acid affords another exception because there is no clear evidence that it is attached covalently to protein, as the definition of a proteoglycan given above specifies. Both GAGs and proteoglycans have proved difficult to work with, partly because of their complexity. However, they are major components of the ground substance; they have a number of important biologic roles; and they are involved in a number of disease processes—so that interest in them is increasing rapidly.
Proteoglycans (mucoproteins) are formed of glycosaminoglycans (GAGs) covalently attached to the core proteins.
They are found in all connective tissues, extracellular matrix (ECM) and on the surfaces of many cell types. Proteoglycans are remarkable for their diversity (different cores, different numbers of GAGs with various lenghts and compositions).
Glycosaminoglycans forming the proteoglycans are the most abundant heteropolisaccharides in the body. They are long unbranched molecules containing a repeating disaccharide unit. Usually one sugar is an uronic acid (either D-glucuronic or L-iduronic) and the other is either GlcNAc or GalNAc. One or both sugars contain sulfate groups (the only exception is hyaluronic acid).
GAGs are highly negatively charged what is essential for their function.
THE SPECIFIC GAGs OF PHYSIOLOGICAL SIGNIFICANCE ARE :
Hyaluronic acid (D-glucuronate + GlcNAc)
Occurence : synovial fluid, ECM of loose connective tissue
Hyaluronic acid is unique among the GAGs because it does not contain any sulfate and is not found covalently attached to proteins. It forms non-covalently linked complexes with proteoglycans in the ECM. Hyaluronic acid polymers are very large (100 - 10,000 kD) and can displace a large volume of water.
Dermatan sulfate (L-iduronate + GlcNAc sulfate)
Occurence : skin, blood vessels, heart valves
Chondroitin sulfate (D-glucuronate + GalNAc sulfate)
Occurence : cartilage, bone, heart valves ; It is the most abundant GAG.
Heparin and heparan sulfate (D-glucuronate sulfate + N-sulfo-D-glucosamine)
Heparans have less sulfate groups than heparins
· Heparin :component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin.
Figure. Structure of heparin. The polymer section illustrates structural features typical of heparin; however, the sequence of variously substituted repeating disaccharide units has been arbitrarily selected. In addition, non-O-sulfated or 3-O-sulfated glucosamine residues may also occur.
· Heparan sulfate: basement membranes, component of cell surfaces.
Keratan sulfate ( Gal + GlcNAc sulfate)
Occurence : cornea, bone, cartilage;
Keratan sulfates are often aggregated with chondroitin sulfates.
The GAGs extend perpendicular from the core protein in a bottlebrush- like structure.
Some forms of keratan sulfates are linked to the protein core through an N-asparaginyl bond.
The protein cores of proteoglycans are rich in Ser and Thr residues which allows multiple GAG attachment.
Proteoglycans Have Numerous Functions
As indicated above, proteoglycans are remarkably complex molecules and are found in every tissue of the body, mainly in the ECM or “ground substance.”
There they are associated with each other and also with the other major structural components of the matrix, collagen and elastin, in quite specific manners. Some proteoglycans bind to collagen and others to elastin.
These interactions are important in determining the structural organization of the matrix. Some proteoglycans (eg, decorin) can also bind growth factors such as TGF-â, modulating their effects on cells. In addition, some of them interact with certain adhesive proteins such as fibronectin and laminin (see above), also found in the matrix. The GAGs present in the proteoglycans are polyanions and hence bind polycations and cations such as Na+ and K+. This latter ability attracts water by osmotic pressure into the extracellular matrix and contributes to its turgor. GAGs also gel at relatively low concentrations. Because of the long extended nature of the polysaccharide chains of GAGs and their ability to gel, the proteoglycans can act as sieves, restricting the passage of large macromolecules into the ECM but allowing relatively free diffusion of small molecules.
Again, because of their extended structures and the huge macromolecular aggregates they often form, they occupy a large volume of the matrix relative to proteins.
SOME FUNCTIONS OF SPECIFIC GAGS & PROTEOGLYCANS
Hyaluronic acid is especially high in concentration in embryonic tissues and is thought to play an important role in permitting cell migration during morphogenesis and wound repair. Its ability to attract water into the extracellular matrix and thereby “loosen it up” may be important in this regard. The high concentrations of hyaluronic acid and chondroitin sulfates present in cartilage contribute to its compressibility (see below).
Chondroitin sulfates are located at sites of calcification in endochondral bone and are also found in cartilage.
They are also located inside certain neurons and may provide an endoskeletal structure, helping to maintain their shape.
Both keratan sulfate I and dermatan sulfate are present in the cornea. They lie between collagen fibrils and play a critical role in corneal transparency. Changes in proteoglycan composition found in corneal scars disappear when the cornea heals. The presence of dermatan
sulfate in the sclera may also play a role in maintaining the overall shape of the eye. Keratan sulfate I is also present in cartilage.
Heparin is an important anticoagulant. It binds with factors IX and XI, but its most important interaction is with plasma antithrombin III. Heparin can also bind specifically to lipoprotein lipase present in capillary walls, causing a release of this enzyme into the circulation.
Certain proteoglycans (eg, heparan sulfate) are associated with the plasma membrane of cells, with their core proteins actually spanning that membrane. In it they may act as receptors and may also participate in the mediation of cell growth and cell-cell communication.
The attachment of cells to their substratum in culture is mediated at least in part by heparan sulfate. This proteoglycan is also found in the basement membrane of the kidney along with type IV collagen and laminin (see above), where it plays a major role in determining the charge selectiveness of glomerular filtration.
Proteoglycans are also found in intracellular locations such as the nucleus; their function in this organelle has not been elucidated. They are present in some storage or secretory granules, such as the chromaffin granules of the adrenal medulla. It has been postulated that they play a role in release of the contents of such granules. The various functions of GAGs are summarized in Table 2.
Table 2. Some functions of glycosaminoglycans and proteoglycans.
ASSOCIATIONS WITH MAJOR DISEASES & WITH AGING
Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM. Tumor cells can induce fibroblasts to synthesize greatly increased amounts of this GAG, thereby perhaps facilitating their own spread. Some tumor cells have less heparan sulfate at their surfaces, and this may play a role in the lack of adhesiveness that these cells display.
The intima of the arterial wall contains hyaluronic acid and chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans. Of these proteoglycans, dermatan sulfate binds plasma low-density lipoproteins.
In addition, dermatan sulfate appears to be the major GAG synthesized by arterial smooth muscle cells. Because it is these cells that proliferate in atherosclerotic lesions in arteries, dermatan sulfate may play an important role in development of the atherosclerotic plaque.
In various types of arthritis, proteoglycans may act as autoantigens, thus contributing to the pathologic features of these conditions. The amount of chondroitin sulfate in cartilage diminishes with age, whereas the amounts of keratan sulfate and hyaluronic acid increase.
These changes may contribute to the development of osteoarthritis. Changes in the amounts of cer tain GAGs in the skin are also observed with aging and help to account for the characteristic changes noted in this organ in the elderly.
An exciting new phase in proteoglycan research is opening up with the findings that mutations that affect individual proteoglycans or the enzymes needed for their synthesis alter the regulation of specific signaling pathways in drosophila and Caenorhabditis elegans, thus affecting development; it already seems likely that similar effects exist in mice and humans.
EXAMPLES OF GAG BINDING PROTEINS:
Secreted proteases and antiproteases
For example antithrombin III (AT III) binds tightly to heparin and certain heparan sulfates (so do its substrates). Thus they control the blood coagulation. In the absence of GAGs AT III inactivates proteases (such as thrombin, factors IXa and XIa) very slowly. In the presence of appropriate GAGs these reactions are accelerated 2000-fold.
GAGs are sufficiently long that both protease and protease inhibitor can bind to the same chain (thus the likelyhood of the two proteins binding to each other is increased enormously). GAGs also affect the protein conformation that contributes to improving AT III binding kinetics.
Polypeptide growth factors
Members of the FGF family, as well as several other growth factors, bind to heparin or heparan sulfate. Binding to endogenous GAGs entraps these molecules in ECM from which they may be later released. GAGs can alter the conformation, proteolytic susceptibility and biological activity of some of these proteins. The bound growth factor is resistant to degradation by extracellular proteases. Active hormone is released by proteolysis of the heparan sulfate chains. It occurs during the tissue growth and remodeling after infection.
Most of the large, multidomain ECM proteins contain at least one GAG binding site.
For example fibrous collagens (type I, III, V) and fibronectin bind to heparan sulfate chains which are attached to the integral membrane core proteins of cell surface proteoglycans such as syndecan and fibroglycan. Cell surface proteoglycans are thought to anchor cells to matrix fibers.
Cell-cell adhesion molecules
· For example NCAM (see cadherins) interacts with cell surface heparan sulfate proteoglycans. This interaction is required for its function. NCAM has a distinct heparan binding domain.
· Hyaluronan is bound to the surface receptors (e.g. CD44) of many migrating cells. It is very important during differentiation (for example myoblasts which are undifferentiated muscle cell precursors bear hyaluronan- rich coat that prevents premature cell fusion). Because its loose, hydrated porous structure, the hyaluronan coat keeps cells apart from each other. They are free to move around and proliferate.
When the level of hyaluronan is lower (e.g. because of digesting by hyaluronidase), there is ceesation of cell movement and initiation of cell- cell attachment.
Mucopolysaccharidoses and collagenoses, their biochemical diagnostics
Mucopolysaccharidoses are a group of metabolic disorders caused by the absence or malfunctioning of lysosomal enzymes needed to break down molecules called glycosaminoglycans - long chains of sugar carbohydrates in each of our cells that help build bone, cartilage, tendons, corneas, skin and connective tissue. Glycosaminoglycans (formerly called mucopolysaccharides) are also found in the fluid that lubricates our joints.
People with a mucopolysaccharidosis disease either do not produce enough of one of the 11 enzymes required to break down these sugar chains into simpler molecules, or they produce enzymes that do not work properly. Over time, these glycosaminoglycans collect in the cells, blood and connective tissues. The result is permanent, progressive cellular damage which affects appearance, physical abilities, organ and system functioning, and, in most cases, mental development.
The mucopolysaccharidoses are part of the lysosomal storage disease family, a group of more than 40 genetic disorders that result when a specific organelle in our body's cells – the lysosome – malfunctions. The lysosome is commonly referred to as the cell’s recycling center because it processes unwanted material into substances that the cell can utilize. Lysosomes break down this unwanted matter via enzymes, highly specialized proteins essential for survival. Lysosomal disorders like mucopolysaccharidosis are triggered when a particular enzyme exists in too small an amount or is missing altogether.
The mucopolysaccharidoses share many clinical features but have varying degrees of severity. These features may not be apparent at birth but progress as storage of glycosaminoglycans affects bone, skeletal structure, connective tissues, and organs. Neurological complications may include damage to neurons (which send and receive signals throughout the body) as well as pain and impaired motor function. This results from compression of nerves or nerve roots in the spinal cord or in the peripheral nervous system, the part of the nervous system that connects the brain and spinal cord to sensory organs such as the eyes and to other organs, muscles, and tissues throughout the body.
Depending on the mucopolysaccharidosis subtype, affected individuals may have normal intellect or have cognitive impairments, may experience developmental delay, or may have severe behavioral problems. Many individuals have hearing loss, either conductive (in which pressure behind the ear drum causes fluid from the lining of the middle ear to build up and eventually congeal), neurosensitive (in which tiny hair cells in the inner ear are damaged), or both. Communicating hydrocephalus — in which the normal reabsorption of cerebrospinal fluid is blocked and causes increased pressure inside the head — is common in some of the mucopolysaccharidoses. Surgically inserting a shunt into the brain can drain fluid. The eye's cornea often becomes cloudy from intracellular storage, and glaucoma and degeneration of the retina also may affect the patient's vision.
Physical symptoms generally include coarse or rough facial features (including a flat nasal bridge, thick lips, and enlarged mouth and tongue), short stature with disproportionately short trunk (dwarfism), dysplasia (abnormal bone size and/or shape) and other skeletal irregularities, thickened skin, enlarged organs such as liver (hepatomegaly) or spleen (splenomegaly), hernias, and excessive body hair growth. Short and often claw-like hands, progressive joint stiffness, and carpal tunnel syndrome can restrict hand mobility and function. Recurring respiratory infections are common, as are obstructive airway disease and obstructive sleep apnea. Many affected individuals also have heart disease, often involving enlarged or diseased heart valves.
Another lysosomal storage disease often confused with the mucopolysaccharidoses is mucolipidosis. In this disorder, excessive amounts of fatty materials known as lipids (another principal component of living cells) are stored, in addition to sugars. Persons with mucolipidosis may share some of the clinical features associated with the mucopolysaccharidoses (certain facial features, bony structure abnormalities, and damage to the brain), and increased amounts of the enzymes needed to break down the lipids are found in the blood.
Seven distinct clinical types and numerous subtypes of the mucopolysaccharidoses have been identified. Although each mucopolysaccharidosis (MPS) differs clinically, most patients generally experience a period of normal development followed by a decline in physical and/or mental function.
Diagnosis often can be made through clinical examination and urine tests (excess mucopolysaccharides are excreted in the urine). Enzyme assays (testing a variety of cells or body fluids in culture for enzyme deficiency) are also used to provide definitive diagnosis of one of the mucopolysaccharidoses. Prenatal diagnosis using amniocentesis and chorionic villus sampling can verify if a fetus either carries a copy of the defective gene or is affected with the disorder. Genetic counseling can help parents who have a family history of the mucopolysaccharidoses determine if they are carrying the mutated gene that causes the disorders.
Any of various diseases or abnormal states (as rheumatoid arthritis, systemic lupus erythematosus, polyarteritis nodosa, rheumatic fever, and dermatomyositis) characterized by inflammatory or degenerative changes in connective tissue—called also collagen disease, collagenolysis, collagen vascular disease
• The major components of the ECM are the structural proteins collagen, elastin, and fibrillin; a number of specialized proteins (eg, fibronectin and laminin); and various proteoglycans.
• Collagen is the most abundant protein in the animal kingdom; approximately 19 types have been isolated.
• Collagen and elastin are fibrous proteins. Collagen molecules contain an abundance of proline, lysine, and glycine, the latter occurring at every third position in the primary structure. Collagen also contains hydroxyproline, hydroxylysine, and glycosylated hydroxylysine, each formed by posttranslational modification.
• Collagen molecules typically form fibrils containing a long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains are wound around one another in a rope-like superhelix (triple helix). Other types of collagen form mesh-like networks.
All collagens contain greater or lesser stretches of triple helix and the repeating structure (Gly-X-Y)n.
• The biosynthesis of collagen is complex, featuring many posttranslational events, including hydroxylation of proline and lysine.
• Diseases associated with impaired synthesis of collagen include scurvy, osteogenesis imperfecta, Ehlers-Danlos syndrome (many types), and Menkes disease.
• Elastin confers extensibility and elastic recoil on tissues.
• Elastin lacks hydroxylysine, Gly-X-Y sequences, triple helical structure, and sugars but contains desmosine and isodesmosine cross-links not found in collagen.
• Elastin is a connective tissue protein with rubber-like properties in tissues such as the lung. 1-Antitrypsin (α1-AT), produced primarily by the liver but also by tissues such as monocytes and alveolar macrophages, prevents elastin degradation in the alveolar walls. A deficiency of α1-AT can cause emphysema and, in some cases, cirrhosis of the liver.
• Fibrillin is located in microfibrils. Mutations in the gene for fibrillin cause Marfan syndrome.
• The glycosaminoglycans (GAGs) are made up of repeating disaccharides containing a uronic acid (glucuronic or iduronic) or hexose (galactose) and a hexosamine (galactosamine or glucosamine). Sulfate is also frequently present.
• The major GAGs are hyaluronic acid, chondroitin 4- and 6-sulfates, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate.
• The GAGs are synthesized by the sequential actions of a battery of specific enzymes (glycosyltransferases, epimerases, sulfotransferases, etc) and are degraded by the sequential action of lysosomal hydrolases. Genetic deficiencies of the latter result in mucopolysaccharidoses (eg, Hurler syndrome).
• GAGs occur in tissues bound to various proteins (linker proteins and core proteins), constituting proteoglycans.
These structures are often of very high molecular weight and serve many functions in tissues.
• Many components of the ECM bind to proteins of the cell surface named integrins; this constitutes one pathway by which the exteriors of cells can communicate with their interiors.
• Bone and cartilage are specialized forms of the ECM.
Collagen I and hydroxyapatite are the major constituents of bone. Collagen II and certain proteoglycans are major constituents of cartilage.