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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 22.4Noncollagen Components of the Extracellular Matrix

In addition to the insoluble fibers of collagen, the extracellular matrix contains two major classes of soluble proteins: multiadhesive matrix proteins, which bind cell-surface adhesion receptors, and proteoglycans, a diverse group of macromolecules containing a core protein with multiple attached polysaccharide chains (see Figure 22-1). Another important component of the matrix, hyaluronan, is a large polysaccharide that forms a highly hydrated gel, making the matrix resilient to compression.

Multiadhesive matrix proteins are long flexible molecules that contain domains responsible for binding a variety of collagen types, other matrix proteins, polysaccharides, cell-surface proteins, and signaling molecules such as growth factors and hormones. Their major role is to attach cells to the extracellular matrix. The importance of these matrix proteins in initiating cellular responses through classic signal-transduction pathways has been recognized in recent years. Both roles are important for organizing the other components of the matrix and also for regulating cell attachment to the matrix, cell migration, and cell shape.

Proteoglycans are found in all connective tissues and extracellular matrices; they also are attached to the surface of many cells. Because of their high content of charged polysaccharides, proteoglycans are highly hydrated. The swelled, hydrated structure of proteoglycans is largely responsible for the volume of the extracellular matrix and also acts to permit diffusion of small molecules between cells and tissues.

We begin our discussion of these matrix components with the multiadhesive proteins because they bind collagen, and then we describe the unique structural features of proteoglycans and hyaluronan.

Laminin and Type IV Collagen Form the Two-Dimensional Reticulum of the Basal Lamina

As we’ve seen already, the basal lamina is a thin sheetlike network of ECM components, usually no more than 60 – 100 nm thick. Most epithelial and endothelial cells rest upon a basal lamina, which is linked to specific plasma-membrane receptor proteins and to fibrous collagens and other components of the underlying loose connective tissue (see Figure 15-23). Individual muscle cells and adipocytes also are surrounded by a basal lamina (Figure 22-18). After type IV collagen, laminin, a large multiadhesive matrix protein, is the most prevalent constituent of all basal laminae. The basal lamina is often called the type IV matrix after its collagen component. All the ECM components are synthesized by cells that rest on the basal lamina.

Figure 22-18. Electron micrograph showing the association of the plasma membrane of skeletal muscle with the basal lamina.

Figure 22-18

Electron micrograph showing the association of the plasma membrane of skeletal muscle with the basal lamina. In this quick-freeze deep-etch preparation, the basal lamina is seen as a meshwork (more...)

The laminins are a family of cross-shaped proteins that are as long as the basal lamina is thick. In adult animals, laminin is a heterotrimeric protein with a total molecular weight of 820,000 (Figure 22-19). Several laminin isoforms, containing slightly different A, B, or C chains, have been identified. Laminin has high-affinity binding sites for other components of the basal lamina, including collagen IV, and for certain cell-adhesion molecules on the surface of many cells. In vitro, laminin molecules assemble into a feltlike web, primarily via interactions between the ends of the arms.

Figure 22-19. Structure of laminin, a large heterotrimeric multiadhesive matrix protein found in all basal laminae.

Figure 22-19

Structure of laminin, a large heterotrimeric multiadhesive matrix protein found in all basal laminae. The cross-shaped molecule contains globular domains and a coiled-coil region in which (more...)

Although laminin and type IV collagen form the basic reticulum of the basal lamina, most basal laminae in adult animals also contain perlecan, a heparan sulfate proteoglycan, and entactin, a small multiadhesive matrix protein that interacts with laminin and type IV collagen. The multiple interactions between these components connect the type IV and laminin networks and stabilize the overall structure of the basal lamina (Figure 22-20). Both type IV collagen and laminin also bind to specific integrins, an important class of cell-adhesion molecules present in the plasma membrane. These interactions attach a basal lamina to adjacent cells.

Figure 22-20. Model of the basal lamina.

Figure 22-20

Model of the basal lamina. [P. D. Yurchenco and J. C. Schittny, 1990, FASEB J. 4: 1577–1590.]

The basal lamina is structured differently in different tissues (Figure 22-21). For instance, the endothelial cells that line capillaries are polarized, with one surface facing the blood. The surface not facing the blood is surrounded by a basal lamina that forms a filter for regulating passage of proteins and other molecules from the blood into the tissues. The basal lamina underlying polarized epithelial cells lining the intestine likewise regulates passage of nutrients into the bloodstream. In smooth muscle, on the other hand, the basal lamina connects adjacent cells and maintains the integrity of the tissue. In the kidney glomerulus, a double-thickness basal lamina separates two cell sheets and acts as a filter in forming the urine.

Figure 22-21. Organization of the basal lamina in different tissues.

Figure 22-21

Organization of the basal lamina in different tissues. (a, b) The basal laminae associated with endothelial cells and muscle cells separate these cells from the underlying or surrounding (more...)

As discussed in Chapter 23, laminin and components of the basal lamina play important roles in embryonic development. For instance, the basal lamina helps four- and eight-celled embryos adhere together in a ball. During development of the nervous system, neurons migrate along extra- cellular-matrix pathways that contain laminin and other matrix components. Thus, the basal lamina not only is important for organizing cells into tissues but for guiding migrating cells during development.

Fibronectins Bind Many Cells to Fibrous Collagens and Other Matrix Components

Fibronectins are another important class of soluble multiadhesive matrix proteins. Their primary role is attaching cells to all matrices that contain the fibrous collagens (types I, II, III, and V). The presence of fibronectin on the surface of nontransformed cultured cells, and its absence on transformed (or tumorigenic) cells, first led to the identification of fibronectin as an adhesive protein. By their attachments, fibronectins regulate the shape of cells and the organization of the cytoskeleton; they are essential for migration and cellular differentiation of many cell types during embryogenesis. Fibronectins also are important for wound healing because they facilitate migration of macrophages and other immune cells into the affected area.

Fibronectins are dimers of two similar polypeptides linked at their C-termini by two disulfide bonds; each chain is about 60 – 70 nm long and 2 – 3 nm thick. At least 20 different fibronectin chains have been identified, all of which are generated by alternative splicing of the RNA transcript of a single fibronectin gene (see Figure 11-24). Analysis of digests of fibronectin with low amounts of proteases shows that the polypeptides consist of six domains. Each domain, in turn, contains repeated sequences that can be classified into one of three types on the basis of similarities in amino acid sequence (Figure 22-22).

The multiadhesive property of fibronectin arises from the presence in different domains of high-affinity binding sites for collagen and other ECM components and for certain integrins on the surface of cells. Proteolytic digestion of the cell-binding domain in one of the type III repeats showed that a segment of about 100 amino acids could bind to integrins. Studies with synthetic peptides corresponding to parts of this segment identified the tripeptide sequence Arg-Gly-Asp (usually abbreviated RGD) as the minimal structure required for recognition by integrins in the plasma membrane. For example, when this tripeptide was covalently linked to an inert protein such as albumin and dried on a culture dish, it stimulated adhesion of fibroblasts to the surface of the dish similar to the effect of intact fibronectin. In the three-dimensional structure of this fibronectin type III repeat, the RGD sequence is at the apex of a loop that protrudes outward from the molecule, in a position to bind to an integrin or other protein (Figure 22-23).

Figure 22-23. Three-dimensional structure of the type III repeat of fibronectin that contains the RGD integrin-binding sequence.

Figure 22-23

Three-dimensional structure of the type III repeat of fibronectin that contains the RGD integrin-binding sequence. The core of seven β strands in this domain has a structure similar (more...)

Although the RGD peptide is the minimal structure required for binding to several integrins, its affinity for integrins is substantially less than that of intact fibronectin or of the entire cell-binding domain. Thus, sequences surround-ing the RGD sequence in fibronectin and other proteins apparently enhance binding to integrins. Further, the affinity of the RGD sequence for integrins is dependent on the absorbed state of fibronectin. Fibronectin in solution or circulating in blood binds to integrins on fibroblasts poorly. Absorption of fibronectin to a surface — in animals to a collagen matrix or the basal lamina surrounding an endothelial cell or, experimentally, to a tissue-culture dish — enhances its ability to bind to cells, probably because the segment of the protein containing the RGD sequence becomes more exposed.

Fibronectin circulating in the blood, which is secreted by the liver, lacks one or both of the type III repeats designated EDA and EDB (for extra domain A and B) present in fibronectin secreted by cultured fibroblasts (see Figure 22-22). Circulating fibronectin forms insoluble matrices somewhat less readily than fibronectin within tissues, but it does bind to fibrin, a constituent of blood clots. Following binding to fibrin, the immobilized fibronectin binds, via its exposed RGD-containing domain, to integrins expressed on passing, activated platelets. As a result, the platelets are localized to damaged regions of blood vessels and can participate in expansion of blood clots. As this example illustrates, both the polymerization of fibronectin into filaments and adhesion of cells to fibronectin are closely controlled to meet the organism’s needs. Later we will return to this topic when we examine the role of fibronectins in cell adhesion and motility.

Proteoglycans Consist of Multiple Glycosaminoglycans Linked to a Core Protein

The proteoglycans have a much higher ratio of polysaccharide to protein than do collagen, fibronectin, and similar glycoproteins in the extracellular matrix. The polysaccharide chains in proteoglycans are long repeating linear polymers of specific disaccharides called glycosaminoglycans (GAGs). Usually one sugar is a uronic acid (either D-glucuronic acid or L-iduronic acid) and the other is either N-acetylglucosamine or N-acetylgalactosamine (Figure 22-24). One or both of the sugars contain one or two sulfate residues. Thus each GAG chain bears many negative charges. Frequently some of the residues in a GAG chain are modified after synthesis; dermatan sulfate is formed from chondroitin sulfate, for instance. Similarly, heparin (used medicinally as an anticlotting drug) is formed, only in mast cells, as a result of enzymatic addition of sulfate groups at specific sites in heparan sulfate. Proteoglycans commonly are named according to the structure of their principal repeating disaccharide in the attached GAGs.

In the synthesis of all proteoglycans, heparan sulfate or chondroitin sulfate chains are formed by the sequential addition of the repeating units to a three-sugar “linker” that is attached to serine residues in a core protein molecule (Figure 22-25). One of the “signal sequences” in a core protein that specifies addition of this linker sugar is Ser-Gly-X-Gly, where X is any amino acid. However, not all such sites in the core protein become substituted, and GAGs are attached to serines in other sequences. Thus the conformation of the core protein may be more important than localized primary sequences in determining where the GAG chains attach. In addition, the mechanisms determining the length of the chains are unknown.

Figure 22-25. Glycosaminoglycan chains in proteoglycans.

Figure 22-25

Glycosaminoglycan chains in proteoglycans. Synthesis of a chondroitin sulfate chain is initiated by transfer of a xylose residue to a serine residue in the core protein, most likely in the (more...)

Proteoglycans, which are remarkable for their diversity, are present both in the extracellular matrix and on the surface of many cells. A given extracellular matrix may contain proteoglycans with several different types of core proteins, and the number, length, and composition of the GAG chains attached to each core protein may vary. Thus, the molecular weight and charge density of a population of proteoglycans can be expressed only as an average; individual molecules can differ considerably. Nonetheless, a good deal is known of the structure and function of certain extracellular and cell-surface proteoglycans.

Extracellular Matrix Proteoglycans

One of the most important extracellular proteoglycans is aggrecan, the predominant proteoglycan in cartilage. As its name implies, aggrecan forms very large aggregates, termed proteoglycan aggregates. A single aggregate, one of the largest macromolecules known, can be more than 4 mm long and have a volume larger than that of a bacterial cell. These aggregates give cartilage its unique gel-like properties and its resistance to deformation, essential for distributing the load in weight-bearing joints.

The central component of the cartilage proteoglycan aggregate is a long molecule of hyaluronan. Bound to it, tightly but noncovalently at 40-nm intervals, are aggrecan core proteins decorated with GAGs (Figure 22-26a). The aggrecan core protein (≈250,000 MW) has one N-terminal globular domain that binds with high affinity to a decasaccharide sequence in hyaluronan; this binding is facilitated by a link protein that binds to the aggrecan core protein and hyaluronan (Figure 22-26b). Covalently attached to each aggrecan core protein, via the trisaccharide linker, are multiple chains of chondroitin sulfate and keratan sulfate. The molecular weight of an aggrecan monomer — that is, the core protein plus the bound glycosaminoglycans — averages 2×106. The entire proteoglycan aggregate, which may contain upward of 100 aggrecan monomers, has a molecular weight in excess of 2×108.

Figure 22-26. Structure of cartilage proteoglycan aggregate.

Figure 22-26

Structure of cartilage proteoglycan aggregate. (a) Electron micrograph of a proteoglycan aggregate from fetal bovine epiphyseal cartilage. Aggrecan core proteins are bound at ≈40-nm (more...)

Not all extracellular proteoglycans form large aggregates like aggrecan. A class of proteoglycans present in the basal lamina, for example, consists of a core protein (20,000 – 40,000 MW) to which are attached several heparan sulfate chains. Such proteoglycans bind to type IV collagen and other structural proteins discussed later, thereby imparting structure to the basal lamina.

Image med.jpgThe importance of the GAG chains that are part of various matrix proteoglycans is illustrated by the rare humans who have a genetic defect in one of the enzymes required for synthesis of dermatan sulfate. These individuals have many defects in their bones, joints, and muscles; do not grow to normal height; and have wrinkled skin, giving them a prematurely aged appearance.

Cell-Surface Proteoglycans

Proteoglycans are attached to the surface of many types of cells, particularly epithelial cells. The most common proteoglycan on the plasma membrane is syndecan, whose general structure is illustrated in Figure 22-27. The core protein of cell-surface proteoglycans spans the plasma membrane and contains a short cytosolic domain, as well as a long external domain to which a small number of heparan sulfate chains are attached; some cell-surface proteoglycans also contain chondroitin sulfate. The GAG chains are attached to serine residues in the core protein via the same trisaccharide linker that is present in extracellular proteoglycans.

Figure 22-27. Schematic diagram of the cell-surface proteoglycan syndecan-4.

Figure 22-27

Schematic diagram of the cell-surface proteoglycan syndecan-4. The core protein in all syndecan proteoglycans (syndecan-1, -2, -3, and -4) spans the plasma membrane and dimerizes (more...)

The extracellular and cytoplasmic domains of syndecan have separate functions. The heparan sulfate chains on the extracellular domain bind to fibrous collagens (types I, III, and IV) and to fibronectin, which are present in the interstitial matrix surrounding the basal lamina. In this way, cell-surface proteoglycans are thought to anchor cells to matrix fibers. Like many integral membrane proteins, the cytoplasmic domain of syndecan interacts with the actin cytoskeleton and in some cases phosphoinositides and protein kinase C. Thus, cell-surface proteoglycans function much like some of the cell-adhesion molecules in the plasma membrane.

Many Growth Factors Are Sequestered and Presented to Cells by Proteoglycans

Besides acting as structural components of the extracellular matrix and anchoring cells to the matrix, both extracellular and cell-surface proteoglycans, particularly those containing heparan sulfate, also bind many protein growth factors (Chapter 20). For instance, fibroblast growth factor (FGF) binds tightly to the heparan sulfate chains in extracellular proteoglycans. Since the bound growth factor is resistant to degradation by extracellular proteases, it serves as a reservoir of matrix-bound FGF (Figure 22-28). Active hormone is released by proteolysis of the proteoglycan core protein or by partial degradation of the heparan sulfate chains, processes that occur during tissue growth and remodeling or after infection. FGF also binds to cell-surface heparan sulfate proteoglycans such as syndecan, which then “present” the bound FGF to its receptor in the plasma membrane, inducing proliferation. Free FGF cannot interact with the FGF receptor, and cells that cannot synthesize heparan sulfate proteoglycans do not respond to FGF. Another example is transforming growth factor β (TGFβ), whose role in embryonic development is discussed in Chapter 23. The core protein of a cell-surface proteoglycan called beta-glycan binds TGFβ and then presents it to TGFβ receptors. These examples illustrate how proteoglycans commonly function as extracellular hormone reservoirs and facilitate binding of hormones to their cell-surface receptors, thus triggering intracellular signaling pathways.

Figure 22-28. Modulation of activity of fibroblast growth factor (FGF) by heparan sulfate proteoglycans.

Figure 22-28

Modulation of activity of fibroblast growth factor (FGF) by heparan sulfate proteoglycans. Free FGF cannot bind to FGF receptors in the plasma membrane. Binding of FGF to heparan sulfate (more...)

Hyaluronan Resists Compression and Facilitates Cell Migration

Hyaluronan (HA), also called hyaluronic acid or hyaluronate, is a major component of the extracellular matrix that surrounds migrating and proliferating cells, particularly in embryonic tissues. It is also a major structural component of the complex proteoglycans that are found in many extracellular matrices, particularly cartilage (see Figure 22-26). Because of its remarkable physical properties, HA imparts stiffness and resilience as well as a lubricating quality to many types of connective tissue such as joints. HA is the only extracellular oligosaccharide that is not covalently linked to a protein.

Each hyaluronan molecule consists of as many as 50,000 repeats of the simple disaccharide glucuronic acid β(1→3) N-acetylglucosamine β(1→4) (see Figure 22-24). If stretched end-to-end, one molecule would be 20 mm long. Individual segments of an HA molecule fold into a stiff rodlike conformation because of the β linkages and extensive intrachain hydrogen bonding between adjacent sugar residues. Mutual repulsion between negatively charged carboxylate groups that protrude outward at regular intervals also contributes to these local rigid structures. Overall, however, HA is not a long, rigid rod as is collagen; rather, in solution it behaves as a random coil about 500 nm in diameter.

Because of the large number of anionic residues on its surface, HA binds a large amount of water and forms, even at low concentrations, a viscous hydrated gel. Given no constraints, an HA molecule will occupy a volume about 1000 times the space of the HA molecule itself. When placed in a confining space, such as in a matrix between two cells, the long HA molecules will tend to push outward. This creates a swelling, or turgor pressure, within the space; the HA molecules push against any fibers or cells that block their motion. Importantly, by binding cations, the COO groups on the surface increase the concentration of ions and thus the osmotic pressure in the HA gel. Large amounts of water are taken up into the matrix, contributing to the turgor pressure within the HA matrix. These swelling forces give connective tissues their ability to resist compression forces, in contrast to collagen fibers, which are able to resist stretching forces.

Hyaluronan is bound to the surface of many migrating cells by a 34-kDa receptor protein termed CD44, or by a homologous protein in the CD44 family. The domain in CD44 that binds HA is similar in sequence and structure to ones found in various extracellular proteoglycans that bind HA. This is one of many examples we shall encounter where a number of different matrix and cell-surface proteins contain domains, or “modules,” of similar structure and function (see Figure 3-10). Almost certainly these arose during evolution from a single ancestral gene that encoded just this domain.

Because of its loose, hydrated, porous nature, the HA “coat” bound to cells appears to keep cells apart from one another, giving them the freedom to move about and proliferate. Cessation of cell movement and initiation of cell-cell attachments are frequently correlated with a decrease in HA, a decrease in the cell-surface molecules that bind HA, and an increase in the extracellular enzyme hyaluronidase, which degrades the matrix HA. These functions of HA are particularly important during the many cell migrations that facilitate differentiation.

SUMMARY

  •  Laminin is a multiadhesive protein in the basal lamina that binds heparan sulfate, type IV collagen, and specific cell-surface receptor proteins.
  •  Fibronectins are multiadhesive proteins that link collagen and other matrix proteins to integrins in the plasma membrane, thereby attaching cells to the matrix.
  •  Glycosaminoglycans are linear chains of 20 – 100 sulfated disaccharides. The most common disaccharides are chondroitin sulfate, heparin and heparan sulfate, and dermatan sulfate (see Figure 22-24).
  •  Proteoglycans consist of multiple glycosaminoglycan chains that branch from a linear protein core. Extracellular proteoglycans are large, highly hydrated molecules that help cushion cells.
  •  In cartilage, a proteoglycan called aggrecan binds at regular intervals to a central hyaluronan molecule, forming a very large aggregate (see Figure 22-26).
  •  Smaller proteoglycans are attached to cell surfaces, where they facilitate cell-matrix interactions and help present certain hormones to their cell-surface receptors (see Figure 22-28).
  •  Hyaluronan is an extremely long, negatively charged polysaccharide. It forms viscous, hydrated gels that resist compression forces. When bound to specific receptors on certain cells, hyaluronan inhibits cell-cell adhesion and facilitates cell migration.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
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