In animals, the extracellular matrix (ECM) has multiple functions. The ECM helps organize cells into tissues and coordinates their cellular functions by activating intracellular signaling pathways that control cell growth, proliferation, and gene expression. The ECM can directly influence cell and tissue structure and function. In addition, it can serve as a repository for inactive or inaccessible signaling molecules (e.g., growth factors) that are released to function when the ECM is disassembled or remodeled by hydrolyases, such as proteases. Indeed, hydrolyzed fragments of ECM macromolecules can themselves have independent biological activity. The ensemble of proteins that compose the ECM itself and associated proteins that covalently mod ify (e.g., chemically cross-link, phosphorylate, cleave), bind to, or otherwise regulate the composition and structure of the ECM is called the matrisome. Proteomic and genomic analyses suggest that there are approximately 1030 and 1110 genes that encode the human and mouse matrisomes, respectively. Dysfunction of matrisome components can cause a wide variety of diseases that affect many different tissues and organs. It is noteworthy that there are ECM components, as well as extracellular domains of plasma membrane proteins, that are phosphorylated on serine, threonine, or tyrosine side chains. Kinases that are present in the luminal compartments of the secretory pathway and some that are apparently secreted into the extracellular space catalyze these phosphorylations.
Many functions of the ECM and, indeed, some features of the assembly of the ECM require transmembrane adhesion receptors, including the integrins, that bind directly to ECM components and that also interact, through adapter proteins, with the cytoskeleton. Adhesion receptors bind to three types of molecules abundant in the ECM of all tissues:
● Proteoglycans, a group of glycoproteins that cushion cells and bind a wide variety of extracellular molecules
● Collagen fibers, which provide structural integrity, mechanical strength, and resilience
● Soluble multi-adhesive matrix proteins, such as laminin and fibronectin, which bind to and cross-link adhesion receptors and other ECM components
The Basal Lamina Provides a Foundation for Assembly of Cells into Tissues
In animals, most organized groups of cells in epithelial and nonepithelial tissues are underlain or surrounded by the basal lamina, a sheet-like meshwork of ECM components usually no more than 60–120 nm thick (Figure 1). The basal lamina is structured differently in different tissues. In columnar and other epithelia such as intestinal lining and skin, it is a foundation on which only one surface of the cells rests. In other tissues, such as muscle or fat, the basal lamina surrounds each cell. Basal laminae play important roles in regeneration after tissue damage and in embryonic development. For instance, the basal lamina helps four- and eight-celled embryos adhere together in a ball. In the development of the nervous system, neurons migrate along ECM pathways that contain basal laminal components. In higher animals, two distinct basal laminae are employed to form a tight barrier that limits diffusion of molecules between the blood and the brain (blood-brain barrier), and in the kidney, a specialized basal lamina serves as a selectively permeable blood filter. In muscle, the basal lamina helps protect the cell membranes from damage during contraction and relaxation. Thus the basal lamina is important for organizing cells into tissues and distinct compartments, repairing tissues, forming permeability barriers, and guiding migrating cells during development. It is therefore not surprising that its components have been highly conserved throughout evolution.
Fig1. A basal lamina separates epithelial cells and some other cells from connective tissue. (a) Transmission electron micrograph of a thin section of cells (top) and underlying connective tissue (bottom). The electron-dense layer of the basal lamina can be seen to follow the undulations of the basal surfaces of the cells. (b) Electron micrograph of a quick-freeze deep-etch preparation of skeletal muscle, showing the plasma membrane, basal lamina, and surrounding connective-tissue collagen fibers. In this preparation, the basal lamina is revealed as a meshwork of filamentous proteins that associates with the plasma membrane and the thicker collagen fibers of the connective tissue. [Part (a) courtesy of Paul Fitzgerald. Part (b) Don W. Fawcett/Science Source.]
Most of the ECM components in the basal lamina are synthesized by the cells that rest on it. Four ubiquitous protein components, each of which comprises multiple, distinct, repeating domains, are found in basal laminae (Figure 2):
●Type IV collagen, trimeric molecules with both rodlike and globular domains that form a two-dimensional network
● Laminins, a family of multi-adhesive, cross-shaped proteins that form a fibrous two-dimensional network with type IV collagen and that also bind to integrins and other adhesion receptors
● Perlecan, a large multidomain proteoglycan that binds to and cross-links many ECM components and cell-surface molecules
● Nidogen (also called entactin), a rodlike molecule that cross-links type IV collagen, perlecan, and laminin, which helps incorporate other components into the ECM and also stabilizes basal laminae.
Fig2. Major protein components of the basal lamina. Type IV collagen and laminin each form two-dimensional networks (see Figures 3 and 5), which are cross-linked by nidogen/entactin and perlecan molecules and which interact via laminins with the plasma membranes of adjacent cells.
Other ECM molecules, such as members of the evolutionarily ancient family of glycoproteins called fibulins, are incorporated into various basal laminae, depending on the tissue and the particular functional requirements of the basal lamina.
One side of the basal lamina is linked to cells by adhesion receptors, including integrins in hemidesmosomes, which bind to laminin in the basal lamina. The other side of the basal lamina is anchored to the adjacent connective tissue by a layer of collagen fibers embedded in a proteoglycan-rich matrix. In stratified squamous epithelia (e.g., skin), this linkage is mediated by anchoring fibrils of type VII collagen. Together, the basal lamina and the anchoring collagen fibrils form the structure called the basement membrane.
Laminin, a Multi-adhesive Matrix Protein, Helps Cross-Link Components of the Basal Lamina
Laminin, the principal multi-adhesive matrix protein in basal laminae, is a heterotrimeric protein comprising α, β, and γ chains. At least 16 laminin isoforms in vertebrates are assembled from 5 α, 3 β, and 3 γ chains, with each chain numbered to reflect the chain composition: laminin-111 (α1β1γ1) or laminin-511 (α5β1γ1). Each laminin isoform exhibits a distinctive pattern of tissue- and developmental stage–specific expression. As shown in Figure 3, many laminins are large, cross-shaped proteins (molecular weight of about 820,000), although some are Y or rod shaped. Globular domains at the N-terminus of each subunit bind to one another and thus mediate the self-assembly of laminins into mesh-like networks. Five globular LG domains at the C-terminus of the laminin α subunit mediate Ca2+-de pendent binding to cell-surface laminin receptors, including certain integrins as well as sulfated glycolipids, syndecan, and dystroglycan. Some of these interactions are via negatively charged carbohydrates on the receptors. LG do mains are found in a wide variety of other proteins and can mediate binding to steroids and proteins as well as carbo hydrates. Laminin is the principal basal laminal ligand of integrins.
Fig3. Laminin is a heterotrimeric multi-adhesive matrix protein found in all basal laminae. (a) Schematic model of cross shaped laminin molecule showing the general shape, location of globular domains, and coiled-coil region in which laminin’s three chains are covalently linked by several disulfide bonds. Different regions of laminin bind to adhesion receptors and various matrix components (indicated by arrows). Right: Laminins assemble into a lattice via interactions between their N-terminal globular domains. See G. R. Martin and R. Timpl, 1987, Annu. Rev. Cell Biol. 3:57; M. Durbeej, 2010, Cell Tissue Res. 339:259–268; and S. Meinen et al., 2007, J. Cell Biol. 176:979–993. (b) Electron micrographs of an intact laminin molecule, showing its characteristic cross shape (left), and the carbohydrate-binding LG do mains near the C-terminus (right). [Part (b) photographs republished with permission of Elsevier, from Timpl, R. et al., “Structure and function of laminin LG modules,” Matrix Biol. 2000, 19(4):309–17; permission conveyed through the Copyright Clearance Center, Inc. Image on right courtesy Jϋrgen Engel.]
Sheet-Forming Type IV Collagen Is a Major Structural Component of the Basal Lamina
Type IV collagen is, together with laminin, a principal structural component of all basal laminae and can bind to adhesion receptors, including some integrins. Collagen IV is one of at least 28 types of collagen in humans that participate in the formation of distinct ECMs in various tissues (Table 1). There are also at least 20 additional collagen-like proteins (such as host defense collagens) in the human proteome. Although collagen isoforms differ in certain structural features and in their tissue distribution, all collagens are trimeric proteins made from three polypeptides, each encoded by one of at least 43 genes in humans, usually called collagen α chains. The three α chains in a collagen molecule can be identical (forming a homotrimer) or different (forming a heterotrimer). All or parts of the three-stranded collagen molecule can twist together into a special triple helix called a collagenous triple helix. When there is more than one triple-helical segment, these segments are joined by nonhelical regions of the protein, as we will see shortly for type IV collagen. Within a helical segment, each of the three α chains twists into a left-handed helix, and the three chains then wrap around one another to form a right-handed triple helix (Figure 4).
Table1. Selected Collagens
Fig4. The collagen triple helix. (a) Left: Side view of the crystal structure of a polypeptide fragment whose sequence is based on repeating sets of three amino acids, Gly-X-Y, characteristic of collagen α chains. Center: Each chain is twisted into a left-handed helix, and three chains wrap around one another to form a right-handed triple helix. The schematic model (right) clearly illustrates the triple-helical nature of the structure and shows the left-handed twist of the individual collagen α chains (red line). (b) View down the axis of the triple helix. The proton side chains of the glycine residues (orange) point into the very narrow space between the polypeptide chains in the center of the triple helix. In collagen mutations in which other amino acids replace glycine, the proton in glycine is replaced by larger groups that disrupt the packing of the chains and destabilize the triple-helical structure. Data from R. Z. Kramer et al., 2001, J. Mol. Biol. 311:131, PDB ID 1bkv.
The collagen triple helix can form because of an unusual abundance of three amino acids in the α chains: glycine, pro line, and a modified form of proline called hydroxyproline. They make up the characteristic repeating sequence motif Gly-X-Y, where X and Y can be any amino acid but are often proline in position X and hydroxyproline in position Y, and less often lysine and hydroxylysine. Glycine is essential because its small side chain, a hydrogen atom, is the only one that can fit into the crowded center of the three stranded helix (see Figure 4b). Hydrogen bonds help hold the three chains together. Although the rigid peptidyl-proline and peptidyl-hydroxyproline linkages are not compatible with formation of a classic single-stranded α helix, they stabilize the distinctive collagenous triple helix. The hydroxyl group in hydroxyproline in the Y position helps hold its ring in a conformation that stabilizes the three-stranded helix.
There are several distinct cell-surface receptors for collagen IV and other types of collagen (other collagens are discussed in the next section). These cell-surface receptors include certain integrins, discoidin domain receptors 1 and 2 (which are tyrosine kinase receptors), glycoprotein VI (on platelets), leukocyte-associated Ig-like receptor-1, members of the mannose receptor family, and a modified form of the protein CD44. They can play critical roles in helping to assemble the ECM and in integrating cellular activity with the ECM.
The unique properties of each collagen isoform are due mainly to differences in (1) the number and lengths of the collagenous triple-helical segments; (2) the segments that flank or interrupt the triple-helical segments and that fold into other kinds of three-dimensional structures; and (3) co valent modification of the α chains (e.g., hydroxylation, glycosylation, oxidation, cross-linking). For example, the chains in type IV collagen are designated IVα chains. Mammals express six homologous IVα chains, which assemble into three different heterotrimeric type IV collagens with distinct properties. All subtypes of type IV collagen, however, form a 400-nm-long triple helix (Figure 5) that is interrupted about 24 times by nonhelical segments and flanked by a large globular domain at the C-terminus of the chain and a smaller globular domain at the N-terminus. The nonhelical regions introduce flexibility into the molecule. Through both lateral associations and interactions entailing the globular N- and C-termini, type IV collagen molecules assemble into a branching, irregular two-dimensional fibrous network that forms a lattice on which, together with the laminin lattice, the basal lamina is built (see Figures 2 and 5).
Fig5. Structure and assembly of type IV collagen. (a) Schematic representation of type IV collagen. This 400-nm-long molecule has a small noncollagenous globular domain at the N-terminus and a large globular domain at the C-terminus. The collagenous triple helix is interrupted by nonhelical segments that introduce flexible kinks into the molecule. Lateral interactions between triple helical segments, as well as head-to-head and tail-to-tail interactions between the globular domains, form dimers, tetramers, and higher order complexes, yielding a sheet-like network. Multiple, unusual sulfilimine (–S=N–) or thioether bonds between hydroxylysine (or lysine) and methionine residues covalently cross-link some adjacent C-terminal domains and contribute to the stability of the network. See A. Boutaud, 2000, J. Biol. Chem. 275:30716. (b) Electron micrograph of type IV collagen network formed in vitro. The lacy appearance results from the flexibility of the molecule, the side-to-side binding between triple-helical segments (white arrows), and the interactions between C-terminal globular domains (yellow arrows). [Part (b) ©1987 Yurchenco, P. D. and Ruben, G. C., J. Cell Biol., 105(6 Pt1):2559–68. doi: 10.1083/ jcb.105.6.2559; Figure 1c.]
In the kidney, a double basal lamina called the glomerular basement membrane separates the epithelium that lines the urinary space from the endothelium that lines the surrounding blood-filled capillaries. Defects in this structure, which is responsible for ultrafiltration of the blood and initial urine formation, can lead to renal failure. For instance, mutations that alter the C-terminal globular domain of certain IVα chains are associated with progressive renal failure as well as sensorineural hearing loss and ocular abnormalities, a condition known as Alport’s syndrome. In Goodpasture’s syndrome, a relatively rare autoimmune dis ease, antibodies bind to the α3 chains of type IV collagen found in the glomerular basement membrane and lungs. This binding sets off an immune response that causes cellular damage, resulting in progressive renal failure and pulmonary hemorrhage.
Perlecan, a Proteoglycan, Cross-Links Components of the Basal Lamina and Cell-Surface Receptors
Perlecan, the major secreted proteoglycan in basal laminae, consists of a large multidomain core protein (∼470 kDa) to which polysaccharides are covalently attached. The core protein is made up of multiple repeats of five distinct domains, including laminin-like LG domains (3 copies), EGF-like domains (12 copies), and Ig domains (22 copies). The many globular repeats give it the appearance of an ap proximately 200-nm-long string of pearls when visualized by electron microscopy; hence the name perlecan. Perlecan contains three types of covalent polysaccharide chains: N-linked chains (see Chapter 14), O-linked chains, and glycosaminoglycans (GAGs). GAGs are long, linear polymers of repeating disaccharides. Glycoproteins containing covalently attached GAG chains are called proteoglycans. Both the protein and the GAG components of perlecan contribute to its ability to incorporate into and define the structure and function of basal laminae. Because its multiple domains and its polysaccharide chains have distinct binding properties, perlecan binds to dozens of other molecules, including other ECM components (e.g., laminin, nidogen/ entactin), cell-surface receptors, and polypeptide growth factors. Simultaneous binding to these molecules results in perlecan-mediated cross-linking. Perlecan can be found in basal laminae and in non–basal laminal ECM. The adhesion receptor dystroglycan can bind perlecan directly, via perlecan’s LG domains, and indirectly, via its binding to laminin. In humans, mutations in the perlecan gene can lead either to dwarfism or to muscle abnormalities, apparently due to dysfunction of the neuromuscular junction that controls muscle firing.
