Collagen and elastin are examples of common, well-characterized fibrous proteins of the extracellular matrix that serve structural functions in the body. For example, collagen and elastin are found as components of skin, connective tissue, blood vessel walls, and sclera and cornea of the eye. Each fibrous protein exhibits special mechanical properties, resulting from its unique structure, which are obtained by combining specific amino acids into regular, secondary structural elements. This is in contrast to globular proteins, whose shapes are the result of complex interactions between secondary, tertiary, and, sometimes, quaternary structural elements.
Collagen is the most abundant protein in the human body. A typical collagen molecule is a long, rigid structure in which three polypeptides (referred to as “? chains”) are wound around one another in a rope-like triple helix (Figure 4.1). Although these molecules are found throughout the body, their types and organization are dictated by the structural role collagen plays in a particular organ. In some tissues, collagen may be dispersed as a gel that gives support to the structure, as in the extracellular matrix or the vitreous humor of the eye. In other tissues, collagen may be bundled in tight, parallel fibers that provide great strength, as in tendons. In the cornea of the eye, collagen is stacked so as to transmit light with a minimum of scattering. Collagen of bone occurs as fibers arranged at an angle to each other so as to resist mechanical shear from any direction.
Figure 4.1.Triple-stranded helix of collagen.
A. Types of collagen
The collagen superfamily of proteins includes more than 25 collagen types, as well as additional proteins that have collagen-like domains. The three polypeptide ? chains are held together by hydrogen bonds between the chains. Variations in the amino acid sequence of the ? chains result in structural components that are about the same size (approximately 1,000 amino acids long), but with slightly different properties. These ? chains are combined to form the various types of collagen found in the tissues. For example, the most common collagen, type I, contains two chains called ?1 and one chain called ?2 (?12?2), whereas type II collagen contains three ?1 chains (?13). The collagens can be organized into three groups, based on their location and functions in the body (Figure 4.2).
Figure 4.2.The most abundant types of collagen.
1. Fibril-forming collagens
Types I, II, and III are the fibrillar collagens, and have the rope-like structure described above for a typical collagen molecule. In the electron microscope, these linear polymers of fibrils have characteristic banding patterns, reflecting the regular staggered packing of the individual collagen molecules in the fibril (Figure 4.3). Type I collagen fibers are found in supporting elements of high tensile strength (for example, tendon and cornea), whereas fibers formed from type II collagen molecules are restricted to cartilaginous structures. The fibers derived from type III collagen are prevalent in more distensible tissues, such as blood vessels.
Figure 4.3.Collagen fibrils at right have a characteristic banding pattern, reflecting the regularly staggered packing of the individual collagen molecules in the fibril
Electron micrograph of collagen: Natural Toxin Research Center. Texas A&M University-Kingsville. Collagen molecule modified from Mathews, C. K., van Holde, K. E. and Ahern, K. G. Biochemistry. 3rd Ed., Addison Wesley Longman, Inc. 2000. Figure 6.13, p.175.
2. Network-forming collagens
Types IV and VII form a three-dimensional mesh, rather than distinct fibrils (Figure 4.4). For example, type IV molecules assemble into a sheet or meshwork that constitutes a major part of basement membranes.
Figure 4.4.Electron micrograph of a polygonal network formed by association of collagen type IV monomers.
modified from Yurchenco, P. D., Birk, D. E., and Mecham, R. P., eds. (1994) Extracellular Matrix Assembly and Structure, Academic Press, San Diego, California
Basement membranes are thin, sheet-like structures that provide mechanical support for adjacent cells, and function as a semipermeable filtration barrier to macromolecules in organs such as the kidney and the lung
3. Fibril-associated collagens
Types IX and XII bind to the surface of collagen fibrils, linking these fibrils to one another and to other components in the extracellular matrix (see Figure 4.2)
B. Structure of collagen
1. Amino acid sequence
Collagen is rich in proline and glycine, both of which are important in the formation of the triple-stranded helix. Proline facilitates the formation of the helical conformation of each ? chain because its ring structure causes “kinks” in the peptide chain. [Note: The presence of proline dictates that the helical conformation of the ? chain cannot be an ? helix.]
Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together. The glycine residues are part of a repeating sequence, ?Gly?X?Y?, where X is frequently proline and Y is often hydroxyproline (but can be hydroxylysine, Figure 4.5). Thus, most of the ? chain can be regarded as a polytripeptide whose sequence can be represented as (?Gly?Pro?Hyp?)333.
Figure 4.5.Amino acid sequence of a portion of the ?1 chain of collagen.
[Note: Hyp is hydroxyproline and Hyl is hydroxylysine.]
2. Triple-helical structure
Unlike most globular proteins that are folded into compact structures, collagen, a fibrous protein, has an elongated, triple-helical structure that places many of its amino acid side chains on the surface of the triple-helical molecule. [Note: This allows bond formation between the exposed R-groups of neighboring collagen monomers, resulting in their aggregation into long fibers.]
3. Hydroxyproline and hydroxylysine
Collagen contains hydroxyproline (hyp) and hydroxylysine (hyl), which are not present in most other proteins. These residues result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains (Figure 4.6). The hydroxylation is, thus, an example of postt ranslational modification (see Hydroxylation). Hydroxyproline is important in stabilizing the triple-helical structure of collagen because it maximizes interchain hydrogen bond formation.
Figure 4.6.Hydroxylation of prolyl residues of pro-? chains of collagen by prolyl hydroxylase.
The hydroxyl group of the hydroxylysine residues of collagen may be enzymatically glycosylated. Most commonly, glucose and galactose are sequentially attached to the polypeptide chain prior to triple-helix formation (Figure 4.7).
Figure 4.7.Synthesis of collagen.
RER = rough endoplasmic reticulum.
C. Biosynthesis of collagen
The polypeptide precursors of the collagen molecule are formed in fibroblasts (or in the related osteoblasts of bone and chondroblasts of cartilage), and are secreted into the extracellular matrix. After enzymic modification, the mature collagen monomers aggregate and become cross-linked to form collagen fibers.
1. Formation of pro-? chains
Collagen is one of many proteins that normally function outside of cells. Like most proteins produced for export, the newly synthesized polypeptide precursors of ? chains (prepro-? chains) contain a special amino acid sequence at their N-terminal ends. This sequence acts as a signal that, in the absence of additional signals, targets the polypeptide being synthesized for secretion from the cell. The signal sequence facilitates the binding of ribosomes to the rough endoplasmic reticulum (RER), and directs the passage of the prepro-? chain into the lumen of the RER. The signal sequence is rapidly cleaved in the RER to yield a precursor of collagen called a pro-? chain (see Figure 4.7).
The pro-? chains are processed by a number of enzymic steps within the lumen of the RER while the polypeptides are still being synthesized (see Figure 4.7). Proline and lysine residues found in the Y-position of the ?Gly?X?Y?sequence can be hydroxylated to form hydroxyproline and hydroxylysine residues. These hydroxylation reactions require molecular oxygen, Fe2+, and the reducing agent vitamin C (ascorbic acid, see Ascorbic Acid (Vitamin C)), without which the hydroxylating enzymes, prolyl hydroxylase and lysyl hydroxylase, are unable to function (see Figure 4.6).
In the case of ascorbic acid deficiency (and, therefore, a lack of prolyl and lysyl hydroxylation), interchain H-bond formation is impaired, as is formation of a stable triple helix. Additionally, collagen fibrils cannot be cross-linked (see below), greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with ascorbic acid deficiency also often show bruises on the limbs as a result of subcutaneous extravasation of blood due to capillary fragility (Figure 4.8).
Figure 4.8.The legs of a 46-year-old man with scurvy
Kronauer and Buhler, Images in Clinical Medicine, The New England Journal of Medicine, June 15, 1995, Vol. 332, No. 24, p. 1611.
Some hydroxylysine residues are modified by glycosylation with glucose or glucosyl-galactose (see Figure 4.7).
4. Assembly and secretion
After hydroxylation and glycosylation, pro-? chains form procollagen, a precursor of collagen that has a central region of triple helix flanked by the nonhelical amino- and carboxyl-terminal extensions called propeptides (see Figure 4.7). The formation of procollagen begins with formation of interchain disulfide bonds between the C-terminal extensions of the pro-? chains. This brings the three ? chains into an alignment favorable for helix formation. The procollagen molecules move through the Golgi apparatus, where they are packaged in secretory vesicles. The vesicles fuse with the cell membrane, causing the release of procollagen molecules into the extracellular space.
5. Extracellular cleavage of procollagen molecules
After their release, the procollagen molecules are cleaved by N- and C-procollagen peptidases, which remove the terminal propeptides, releasing triple-helical tropocollagen molecules.
6. Formation of collagen fibrils
Individual tropocollagen molecules spontaneously associate to form collagen fibrils. They form an ordered, overlapping, parallel array, with adjacent collagen molecules arranged in a staggered pattern, each overlapping its neighbor by a length approximately three-quarters of a molecule (see Figure 4.7).
7. Cross-link formation
The fibrillar array of collagen molecules serves as a substrate for lysyl oxidase. This Cu2+-containing extracellular enzyme oxidatively deaminates some of the lysyl and hydroxylysyl residues in collagen. The reactive aldehydes that result (allysine and hydroxyallysine) can condense with lysyl or hydroxylysyl residues in neighboring collagen molecules to form covalent cross-links and, thus, mature collagen fibers (Figure 4.9).
Figure 4.9.Formation of cross-links in collagen.
Lysyl oxidase is one of several copper-containing enzymes. Others include cytochrome oxidase (see Cytochrome a + a3), dopamine hydroxylase (see Synthesis of catecholamines), superoxide dismutase (see Enzymes that catalyze antioxidant reactions) and tyrosinase (see Albinism). Disruption in copper homeostasis causes copper deficiency (X-linked Menkes disease) or overload (Wilson disease).
D. Degradation of collagen
Normal collagens are highly stable molecules, having half-lives as long as several years. However, connective tissue is dynamic and is constantly being remodeled, often in response to growth or injury of the tissue. Breakdown of collagen fibers is dependent on the proteolytic action of collagenases, which are part of a large family of matrix metalloproteinases. For type I collagen, the cleavage site is specific, generating three-quarter and one-quarter length fragments. These fragments are further degraded by other matrix proteinases to their constituent amino acids.
E. Collagen diseases: Collagenopathies
Defects in any one of the many steps in collagen fiber synthesis can result in a genetic disease involving an inability of collagen to form fibers properly and, thus, provide tissues with the needed tensile strength normally provided by collagen. More than 1,000 mutations have been identified in 22 genes coding for 12 of the collagen types. The following are examples of diseases that are the result of defective collagen synthesis.
1. Ehlers-Danlos syndrome (EDS)
This disorder is a heterogeneous group of generalized connective tissue disorders that result from inheritable defects in the metabolism of fibrillar collagen molecules. EDS can result from a deficiency of collagen-processing enzymes (for example, lysyl hydroxylase or procollagen peptidase), or from mutations in the amino acid sequences of collagen types I, III, or V. The most clinically important mutations are found in the gene for type III collagen.
Collagen containing mutant chains is not secreted, and is either degraded or accumulated to high levels in intracellular compartments. Because collagen type III is an important component of the arteries, potentially lethal vascular problems occur. [Note: Although collagen type III is only a minor component of the collagen fibrils in the skin, patients with EDS also show, for unknown reasons, defects in collagen type I fibrils. This results in fragile, stretchy skin and loose joints (Figure 4.10).]
Figure 4.10.Stretchy skin of Ehlers-Danlos syndrome.
Photo from Web site Derma.de
2. Osteogenesis imperfecta (OI)
This disease, known as brittle bone syndrome, is also a heterogeneous group of inherited disorders distinguished by bones that easily bend and fracture (Figure 4.11). Retarded wound healing and a rotated and twisted spine leading to a “humped-back” (kyphotic) appearance are common features of the disease. Type I OI is called osteogenesis imperfecta tarda. The disease is the consequence of decreased production of ?1 and ?2 chains. It presents in early infancy with fractures secondary to minor trauma, and may be suspected if prenatal ultrasound detects bowing or fractures of long bones.
Type II OI is called osteogenesis imperfecta congenita, and is the most severe. Patients die of pulmonary hypoplasia in utero or during the neonatal period. Most patients with severe OI have mutations in the gene for either the pro-?1 or pro-?2 chains of type I collagen. The most common mutations cause the replacement of glycine residues (in ?Gly?X?Y?) by amino acids with bulky side chains. The resultant structurally abnormal pro-? chains prevent the formation of the required triple-helical conformation.
Figure 4.11.Lethal form (type II) of osteogenesis mperfecta in which the fractures appear in utero, as revealed by his radiograph of a stillborn fetus.
Jorde, L. B., Carey, J. C., Bamshad, M. J. and White, R. L. Medical Genetics, 2nd Ed.
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 hydroxylysine. 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 polypeptides 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.]
Figure 4.12.Desmosine cross-link in elastin.
Figure 4.13.Elastin fibers in relaxed and stretched conformations.
B. Role of ?1-antitrypsin in elastin degradation
Blood and other body fluids contain a protein, ?1-antitrypsin (?1-AT, A1AT, currently also called ?1-antiproteinase), that inhibits a number of proteolytic enzymes (also called proteases or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was originally named ?1-antitrypsin because it inhibits the activity of trypsin (a proteolytic enzyme synthesized as trypsinogen by the pancreas, see Activation of zymogens.]
?1-AT comprises more than 90% of the ?1-globulin fraction of normal plasma. ?1-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 ?1-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.
Figure 4.14.Destruction of alveolar tissue by elastase released from neutrophils activated as part of the immune response to airborne pathogens.
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 ?1-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 ?1-AT. A number of different mutations in the gene for ?1-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 ?1-AT by the liver. The accumulated polymer may result in cirrhosis (scarring of the liver).
In the United States, the ?1-AT mutation is most common in Caucasians of Northern European ancestry. An individual must inherit two abnormal ?1-AT alleles to be at risk for the development of emphysema. In a heterozygote, with one normal and one defective gene, the levels of ?1-AT are sufficient to protect the alveoli from damage. [Note: A specific ?1-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 ?1-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 ?1-AT. The ?1-AT diffuses from the blood into the lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial cells.
Collagen and elastin are fibrous proteins (Figure 4.15). 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. Elastin is a connective tissue protein with rubber-like properties in tissues such as the lung. ?1-Antitrypsin (?1-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 ?1-AT can cause emphysema and, in some cases, cirrhosis of the liver.
Figure 4.15.Key concept map for the fibrous proteins, collagen and elastin.