Fibrillar Collagens Are the Major Fibrous Proteins in the Extracellular Matrix of Connective Tissues
Connective tissue, such as tendon and cartilage, differs from other solid tissues in that most of its volume is made up of extracellular matrix rather than cells. This matrix is packed with insoluble protein fibers and contains proteoglycans, various multiadhesive proteins, and hyaluronan, a very large, nonsulfated GAG. The most abundant fibrous protein in connective tissue is collagen. Rubberlike elastin fibers, which can be stretched and relaxed, also are present in deformable sites (e.g., skin, tendons, heart). As discussed later, the fibronectins, a family of multiadhesive matrix proteins, form their own distinct fibrils in the matrix of some connective tissues. Although several types of cells are found in connective tissues, the various ECM components are produced largely by cells called fibroblasts.
About 80-90 percent of the collagen in the body consists of types I, II, and III collagens, located primarily in connective tissues. Because of its abundance in tendon-rich tissue such as rat tail, type I collagen is easy to isolate and was the first collagen to be characterized. Its fundamental structural unit is a long (300-nm), thin (1.5-nm-diameter) triple helix consisting of two a 1(I) chains and one a2(I) chain, each precisely 1050 amino acids in length (see Figure 6-14). The triple-stranded molecules associate into higher-order poly mers called collagen fibrils, which in turn often aggregate into larger bundles called collagen fibers.
The minor classes of collagen include fibril-associated collagens, which link the fibrillar collagens to one another or to other ECM components; sheet-forming and anchoring collagens, which form two-dimensional networks in basal laminae (type IV) and connect the basal lamina in skin to the underlying connective tissue (type VII); transmembrane collagens, which function as adhesion receptors; and host defense collagens, which help the body recognize and eliminate pathogens. Table 6-1 lists specific examples in the various classes of collagens. Interestingly, several collagens (e.g., types XVIII and XV) function as core proteins in proteoglycans.
Formation of Collagen Fibrils Begins in the Endoplasmic Reticulum and Is Completed Outside the Cell
Collagen biosynthesis and secretion follow the normal pathway for a secreted protein, which is described in detail in Chapters 16 and 17. The collagen a chains are synthesized as longer precursors, called pro-a chains, by ribosomes attached to the endoplasmic reticulum (ER). The pro-a chains undergo a series of covalent modifications and fold into triple-helical procollagen molecules before their release from cells (Figure 6-20).
I * 7| Propeptide ^ cleavage
Cross-striations (67 nm)
I * 7| Propeptide ^ cleavage
^ Fibril assembly and crosslinking
^ Fibril assembly and crosslinking
Cross-striations (67 nm)
4 FIGURE 6-20 Major events in biosynthesis of fibrillar collagens. Step 1: Procollagen a chains are synthesized on ribosomes associated with the endoplasmic reticulum (ER) membrane, and asparagine-linked oligosaccharides are added to the C-terminal propeptide. Step 2|: Propeptides associate to form trimers and are covalently linked by disulfide bonds, and selected residues in the Gly-X-Y triplet repeats are covalently modified [certain prolines and lysines are hydroxylated, galactose (Gal) or galactose-glucose (hexagons) is attached to some hydroxylysines, prolines are cis ^ trans isomerized]. Step 3: The modifications facilitate zipperlike formation, stabilization of triple helices, and binding by the chaperone protein Hsp47 (Chapter 16), which may stabilize the helices or prevent premature aggregation of the trimers or both. Steps 4 and 5: The folded procollagens are transported to and through the Golgi apparatus, where some lateral association into small bundles takes place. The chains are then secreted (step 6 ),the N- and C-terminal propeptides are removed (step 7), and the trimers assemble into fibrils and are covalently cross-linked (step 8). The 67-nm staggering of the trimers gives the fibrils a striated appearance in electron micrographs (inset). [Adapted from A. V. Persikov and B. Brodsky, 2002, Proc. Nat'l. Acad. Sci. USA 99(3):1101-1103.]
After the secretion of procollagen from the cell, extracellular peptidases (e.g., bone morphogenetic protein-1) remove the N-terminal and C-terminal propeptides. In regard to fibrillar collagens, the resulting molecules, which consist almost entirely of a triple-stranded helix, associate laterally to generate fibrils with a diameter of 50-200 nm. In fibrils, adjacent collagen molecules are displaced from one another by 67 nm, about one-quarter of their length. This staggered array produces a striated effect that can be seen in electron micrographs of collagen fibrils (see Figure 6-20, inset). The unique properties of the fibrous collagens (e.g., types I, II, III) are mainly due to the formation of fibrils.
Short non-triple-helical segments at either end of the collagen a chains are of particular importance in the formation of collagen fibrils. Lysine and hydroxylysine side chains in these segments are covalently modified by extracellular lysyl oxidases to form aldehydes in place of the amine group at the end of the side chain. These reactive aldehyde groups form covalent crosslinks with lysine, hydroxylysine, and histidine residues in adjacent molecules. These cross-links stabilize the side-by-side packing of collagen molecules and generate a strong fibril. The removal of the propeptides and covalent cross-linking take place in the extracellular space to prevent the potentially catastrophic assembly of fibrils within the cell.
0The post-translational modifications of pro-a chains are crucial for the formation of mature collagen molecules and their assembly into fibrils. Defects in these modifications have serious consequences, as ancient mariners frequently experienced. For example, ascorbic acid (vitamin C) is an essential cofactor for the hydroxy-lases responsible for adding hydroxyl groups to proline and lysine residues in pro-a chains. In cells deprived of ascorbate, as in the disease scurvy, the pro-a chains are not hydroxy-lated sufficiently to form stable triple-helical procollagen at normal body temperature, and the procollagen that forms cannot assemble into normal fibrils. Without the structural support of collagen, blood vessels, tendons, and skin become fragile. Because fresh fruit in the diet can supply sufficient vitamin C to support the formation of normal collagen, early British sailors were provided with limes to prevent scurvy, leading to their being called "limeys."
Rare mutations in lysyl hydroxylase genes cause Bruck syndrome and one form of Ehlers-Danlos syndrome. Both disorders are marked by connective-tissue defects, although their clinical symptoms differ. I
Type I and II Collagens Form Diverse Structures and Associate with Different Nonfibrillar Collagens
Collagens differ in their ability to form fibers and to organize the fibers into networks. In tendons, for instance, long type I collagen fibrils are packed side by side in parallel bundles, forming thick collagen fibers. Tendons connect muscles to bones and must withstand enormous forces. Because type
I collagen fibers have great tensile strength, tendons can be stretched without being broken. Indeed, gram for gram, type I collagen is stronger than steel. Two quantitatively minor fibrillar collagens, type V and type XI, coassemble into fibers with type I collagen, thereby regulating the structures and properties of the fibers. Incorporation of type V collagen, for example, results in smaller-diameter fibers.
Type I collagen fibrils are also used as the reinforcing rods in the construction of bone. Bones and teeth are hard and strong because they contain large amounts of dahllite, a crystalline calcium- and phosphate-containing mineral. Most bones are about 70 percent mineral and 30 percent protein, the vast majority of which is type I collagen. Bones form when certain cells (chondrocytes and osteoblasts) secrete collagen fibrils that are then mineralized by deposition of small dahllite crystals.
In many connective tissues, type VI collagen and proteo-glycans are noncovalently bound to the sides of type I fibrils and may bind the fibrils together to form thicker collagen fibers (Figure 6-21a). Type VI collagen is unusual in that the molecule consists of a relatively short triple helix with glob-
▲ FIGURE 6-21 Interactions of fibrous collagens with nonfibrous fibril-associated collagens. (a) In tendons, type I fibrils are all oriented In the direction of the stress applied to the tendon. Proteoglycans and type VI collagen bind noncovalently to fibrils, coating the surface. The microfibrils of type VI collagen, which contain globular and triple-helical segments, bind to type I fibrils and link them together into thicker fibers. (b) In cartilage, type IX collagen molecules are covalently bound at regular intervals along type II fibrils. A chondroitin sulfate chain, covalently linked to the a2(IX) chain at the flexible kink, projects outward from the fibril, as does the globular N-terminal region. [Part (a), see R. R. Bruns et al., 1986, J. Cell Biol. 103:393. Part (b), see L. M. Shaw and B. Olson, 1991, Trends Biochem. Sci. 18:191.]
ular domains at both ends. The lateral association of two type VI monomers generates an "antiparallel" dimer. The end-to-end association of these dimers through their globular domains forms type VI "microfibrils." These microfibrils have a beads-on-a-string appearance, with about 60-nm-long triple-helical regions separated by 40-nm-long globular domains.
The fibrils of type II collagen, the major collagen in cartilage, are smaller in diameter than type I fibrils and are oriented randomly in a viscous proteoglycan matrix. The rigid collagen fibrils impart a strength and compressibility to the matrix and allow it to resist large deformations in shape. This property allows joints to absorb shocks. Type II fibrils are cross-linked to matrix proteoglycans by type IX collagen, another fibril-associated collagen. Type IX collagen and several related types have two or three triple-helical segments connected by flexible kinks and an N-terminal globular segment (Figure 6-22b). The globular N-terminal segment of type IX collagen extends from the fibrils at the end of one of its helical segments, as does a GAG chain that is sometimes linked to one of the type IX chains. These protruding nonhelical structures are thought to anchor the type II fibril to proteoglycans and other components of the matrix. The interrupted triple-helical structure of type IX and related collagens prevents them from assembling into fibrils, although they can associate with fibrils formed from other collagen types and form covalent cross-links to them.
0 Certain mutations in the genes encoding collagen al(I) or a2(I) chains, which form type I collagen, lead to osteogenesis imperfecta, or brittle-bone disease. Because every third position in a collagen a chain must be a glycine for the triple helix to form (see Figure 6-14), mutations of glycine to almost any other amino acid are deleterious, resulting in poorly formed and unstable helices. Only one defective a chain of the three in a collagen molecule can disrupt the whole molecule's triple-helical structure and function. A mutation in a single copy (allele) of either the a1(I) gene or the a2(I) gene, which are located on nonsex chromosomes (autosomes), can cause this disorder. Thus it normally shows autosomal dominant inheritance (Chapter 9). I
Hyaluronan Resists Compression and Facilitates Cell Migration
Hyaluronan, also called hyaluronic acid (HA) or hy-aluronate, is a nonsulfated GAG formed as a disaccharide repeat composed of glucuronic acid and A-acetylglucosamine (see Figure 6-17a) by a plasma-membrane-bound enzyme (HA synthase) and is directly secreted into the extracellular space. It is a major component of the extracellular matrix that surrounds migrating and proliferating cells, particularly in embryonic tissues. In addition, as will be described shortly, hyaluronan forms the backbone of complex proteoglycan aggregates found in many extracellular matrices, particularly cartilage. Because of its remarkable physical properties, hyaluronan imparts stiffness and resilience as well as a lu bricating quality to many types of connective tissue such as joints.
Hyaluronan molecules range in length from a few disac-charide repeats to «25,000. The typical hyaluronan in joints such as the elbow has 10,000 repeats for a total mass of 4 X 106 Da and length of 10 pm (about the diameter of a small cell). Individual segments of a hyaluronan molecule fold into a rodlike conformation because of the p glycosidic linkages between the sugars and extensive intrachain hydrogen bonding. Mutual repulsion between negatively charged carboxylate groups that protrude outward at regular intervals also contributes to these local rigid structures. Overall, however, hyaluronan is not a long, rigid rod as is fibrillar collagen; rather, in solution it is very flexible, bending and twisting into many conformations, forming a random coil.
Because of the large number of anionic residues on its surface, the typical hyaluronan molecule binds a large amount of water and behaves as if it were a large hydrated sphere with a diameter of «500 nm. As the concentration of hyaluronan increases, the long chains begin to entangle, forming a viscous gel. Even at low concentrations, hyaluro-nan forms a hydrated gel; when placed in a confining space, such as in a matrix between two cells, the long hyaluronan molecules will tend to push outward. This outward pushing creates a swelling, or turgor pressure, within the extracellular space. In addition, the binding of cations by COO~ groups on the surface of hyaluronan increases the concentration of ions and thus the osmotic pressure in the gel. As a result, large amounts of water are taken up into the matrix, contributing to the turgor pressure. These swelling forces give connective tissues their ability to resist compression forces, in contrast with collagen fibers, which are able to resist stretching forces.
Hyaluronan is bound to the surface of many migrating cells by a number of adhesion receptors (e.g., one called CD44) containing HA-binding domains, each with a similar three-dimensional conformation. Because of its loose, hydrated, porous nature, the hyaluronan "coat" bound to cells appears to keep cells apart from one another, giving them the freedom to move about and proliferate. The cessation of cell movement and the initiation of cell-cell attachments are frequently correlated with a decrease in hyaluronan, a decrease in HA-binding cell-surface molecules, and an increase in the extracellular enzyme hyaluronidase, which degrades hyaluronan in the matrix. These functions of hyaluronan are particularly important during the many cell migrations that facilitate differentiation and in the release of a mammalian egg cell (oocyte) from its surrounding cells after ovulation.
Association of Hyaluronan and Proteoglycans Forms Large, Complex Aggregates
The predominant proteoglycan in cartilage, called aggrecan, assembles with hyaluronan into very large aggregates, illustrative of the complex structures that proteoglycans sometimes form. The backbone of the cartilage proteoglycan aggregate is a long molecule of hyaluronan to which multiple aggrecan molecules are bound tightly but noncovalently (Figure 6-22a). A single aggrecan aggregate, one of the largest macromolecular complexes known, can be more than 4 mm long and have a volume larger than that of a bacterial cell.
■ N-terminal Hyaluronan-binding domain
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