The collagens are a family of connective tissue proteins characterized by the presence of three polypeptides called alpha chains, which contain molecular domains that are wound together in a ropelike super helix. Collagens are rich in the amino acids proline and glycine, which play roles in the formation and stability of the triple-stranded super helix. The reader is referred to two reviews on collagen biochemistry.^1,2

The genes of at least 28 distinct collagen types have been characterized.³ The interstitial collagens, types I, II, III, and V, exist as large extended molecules that tend to organize into fibrils that may be heterotypic¹—that is, there may be more than one collagen type within these fibrils.⁴ Type IV collagen, also termed basement membrane (BM) collagen, does not exist in fibrillar form but is in a complex network of collagen molecules linked by disulfide and other cross-linkages and associated with noncollagenous molecules, such as LM, entactin, and proteoglycans, to form an amorphous matrix.^5,6 Although at least 28 collagen types are recognized, the protein of only the first 11 collagens has been isolated from tissues.

Table 19-1 presents a summary of the collagen family (a modification of the one reported by Canty and Kadler³). There are 46 genes corresponding to the alpha chains of 28 collagen types. Collagen type I is the most abundant collagen and protein in the body. The basic unit of the type I collagen fibril is a triple helical heterotrimer, tropocollagen, consisting of two identical chains, alpha 1(I), and a third chain, alpha 2(I).¹ The other collagen types have been given similar designations; however, some of the types are homotrimers containing three identical chains and some contain three genetically distinct chains.

The presence of glycine in every third position, along with the extensive hydrogen bonding, provides the triple helix with a compact protected structure resistant to the action of most proteases. The alpha chains of the collagen superfamily are encoded with information that specifies self-assembly into fibrils, microfibrils, and networks that have diverse functions in the ECM.⁶ The structures of collagens can be stabilized further through the formation of covalent cross-linkages derived from modification and condensation of certain lysine and hydroxylysine residues on adjacent alpha chains.² Cross-linkage formation is important in stabilizing collagen fibrils and contributes to their high tensile strength, equivalent to that of fine steel wire.

Elastin

The biochemistry and molecular biology of elastin have been subjects of excellent reviews.^12,13 As in interstitial collagens, glycine makes up about one third of the amino acid content of elastin. Unlike collagen, however, glycine is not present in every third position. In addition, elastin is an exceedingly hydrophobic protein, with a large content of valine, leucine, and isoleucine.

Elastin is synthesized as a precursor molecule, tropoelastin, with a molecular weight of about 70 kDa. However, in tissues, elastin is found as an amorphous macromolecular network. This is because of the condensation of tropoelastin molecules through the formation of covalent cross-linkages unique to elastin. These cross-linkages arise through the condensation of four lysine residues on different tropoelastin molecules to form the cross-linking amino acids, desmosine and isodesmosine, that are characteristic of tissue elastin. The reader is referred to reviews by Bailey and associates² and Wagenseil and Mecham¹² for a more detailed discussion of collagen and elastin cross-linking.

The hydrophobicity, together with the formation of cross-linkages, endow elastin with its elastic properties as well as its extreme insolubility and amorphous structure. Elastin accounts for most of the elastic properties of skin, arteries, ligaments, and the lungs. The presence of elastin has been demonstrated in other organs, such as the eye and kidney. In most tissues, elastin is found in association with microfibrils, which contain several glycoproteins, including fibrillin. Microfibrils have been identified in many tissues and organs, and the importance of their assembly as determinants of connective tissue architecture has been brought into focus by the identification of mutations in fibrillin in the heritable connective tissue disorder, Marfan syndrome.¹³

An elegant review has summarized knowledge of the structure of the elastin gene, including consideration of the heterogeneity observed in immature mRNA due to alternative splicing in the primary transcript.¹⁴ Analyses of the bovine and human elastin genes have revealed the separation of those exons coding for distinct hydrophobic and cross-linking domains. Comparison of the cDNA and genomic sequences, as well as S1 analyses, have demonstrated that the primary transcript of both species is subject to considerable alternative splicing. It is likely that this accounts for the presence of multiple tropoelastins found in several species. It has been suggested that the differences in alternative splicing may be correlated with aging.¹⁴

Proteoglycans

Proteoglycans are characterized by the presence of highly negatively charged, polymeric chains (glycosaminoglycans [GAGs]) of repeating disaccharide units covalently attached to a core protein. The disaccharide units comprise an N-conjugated amino sugar, either glucosamine or galactosamine, and a uronic acid, usually D-glucuronic acid or, in the case of dermatan sulfate, heparan sulfate, and heparin, L-iduronic acid. In cartilage and in the cornea, another GAG, keratan sulfate, containing D-glucose instead of a uronic acid, has been demonstrated. The amino group of the hexosamine component is generally acetylated, and the GAGs are usually O-sulfated in hexosamine residues with some N-sulfation, instead of acetylation, in the case of heparan sulfate and heparin. Depending on the source and type of proteoglycan, the number of GAGs attached to the core protein can vary from three or four to more than 20, with each GAG having a molecular size in the tens of thousands of daltons. In addition, as in the case of the cartilage proteoglycans, there may be more than one type of GAG attached to the core protein. In cartilage, several proteoglycan molecules may be associated with another very large GAG, hyaluronic acid, consisting of disaccharide units of glucuronyl N-acetylglucosamine. The compositional structure of the GAGs is summarized in Table 19-2.

The overall effect of these structures is the creation of huge, negatively charged highly hydrophobic complexes. The hydration and charge properties of these complexes cause them to become highly extended, occupying a hydrodynamic volume in the tissue much larger than that which would be predicted from their chemical composition. In the case of synovial cartilage, it is suggested that the hydration endows the tissue with shock-absorbing properties in which applied pressure to the joint is counteracted by the extrusion of water from the complex, forcing a compression of the negative charges within the molecule. On the release of pressure, the electronegative repulsive forces drive the charges apart, with a concomitant influx of water to restore the initial hydrated state. The metachromatic staining properties of connective tissues are mainly because of their proteoglycan content. There have been several excellent reviews of proteoglycan biochemistry.^15–17

In recent years, several proteoglycans have been identified in the pericellular environment, associated with cell surfaces or interacting with ECM components, such as interstitial collagens, FN, and transforming growth factor-β (TGF-β). Reviews by Groffen and coworkers¹⁵ and Schaefer and Iozzo^16,17 have described the structures of the protein cores and their gene organization, functional characteristics, and tissue distribution. Table 19-3 (a modification of that published by Schaefer and Iozzo¹⁶) lists the biologic characteristics of pericellular proteoglycans. Several of the proteoglycans on the list constitute a group of small, leucine-rich proteoglycans (SLRPs). Notable among them are decorin¹⁷ and perlecan.¹⁸ They are multidomain assemblies of protein motifs with relatively elongated and highly glycosylated structures and have several protein domains shared with other proteins. In their review, Groffen and colleagues¹⁵ discussed the role of perlecan as a crucial determinant of glomerular BM permselectivity and suggested that the additional presence of agrin, another heparan sulfate proteoglycan species, makes the latter important contributors to glomerular function.

Lumican, one of the leucine-rich proteoglycans, is found in relative abundance in articular cartilage,¹⁷ which, along with its size, varies with age. In adult cartilage extracts, it exhibits a molecular size in the range of 55 to 80 kDa. Extracts from juvenile cartilage have a more restricted size variation corresponding to the higher molecular size range present in the adult. In the neonate, the sizes are in the range of 70 to 80 kDa.

The biosynthesis of proteoglycans begins with the synthesis of the core protein. The sugars of the GAG chain, in most cases, are sequentially added to serine residues of the protein using uridine diphosphate conjugates of the component sugars, with sulfation following as the chain elongates. Most of the chain elongation and sulfation is associated with the Golgi apparatus. The degradation of proteoglycans is mediated through the action of lysosomal glycosidases and sulfatases specific for the hydrolysis of the various structural sites within the GAG chain. Genetic abnormalities in the production or synthesis of these enzymes have been shown to be the main causes of the mucopolysaccharidoses, whose victims may exhibit severe tissue abnormalities and a high incidence of mental retardation.

Structural Glycoproteins

In addition to the collagen and elastin components of connective tissues, there are groups of glycoproteins, the structural glycoproteins, that have important roles in the physiology and structural properties of connective and other types of tissues. These proteins, which include FN, LM, entactin-nidogen, thrombospondin (TSP), and others, are involved during development, in cell attachment and spreading, and in tissue growth and turnover.

Fibronectin

One of the best characterized of the structural glycoproteins is fibronectin. It was originally isolated from serum, where it was referred to as cold-insoluble globulin (CIG). As it became recognized that FN was an important secretory product of fibroblasts and other types of cells, and was involved in cell adhesion, the term fibronectin replaced CIG. Comprehensive reviews on the structure and function of FN have been published by Haranuga and Yamada²⁰ and Schwarzbauer and DeSimone.²¹

FN exists as a disulfide-linked dimer with a molecular weight of about 450 kDa, with each monomer having a molecular size of 250 kDa. FN exists in at least two forms, a cell-associated form and a plasma form. Plasma FN is synthesized by hepatocytes and secreted into the circulation. It is somewhat smaller and more soluble at a physiologic pH than the cellular form. Spectrophotometric and ultracentrifugal studies have indicated that both forms are elongated molecules composed of structured domains separated by flexible, extensible regions. Limited proteolytic digestion studies have revealed the presence of specific binding sites for a number of ligands, including collagen, fibrin, cell surfaces, heparin (heparan sulfate proteoglycan), factor XIIIa, and actin.

FN plays a role in blood clotting by becoming cross-linked to fibrin through the action of factor XIIIa transamidase, which catalyzes the final step in the clotting cascade.²² Fibroblasts and other cell types involved in the repair of injury adhere to the clot by interacting with the cell-binding domain of FN. FN contains a unique peptide sequence, arginyl-glycyl-aspartyl-serine (RGDS, RGD), which binds to specific cell surface proteins (integrins) that span the plasma membrane.²¹ Purified RGD can inhibit FN from binding the cells and can even displace bound FN. The integrins have a complex molecular organization and appear to interact with certain intracellular proteins, thereby providing a mechanism for the control of a number of events by components of the extracellular environment.

FN is encoded by a single gene, and its complete primary structure has been determined by the DNA sequencing of overlapping complementary DNA (cDNA) clones.²³ From such studies, it became recognized that there are peptide segments derived from alternative splicing of FN mRNA at three distinct regions, termed extradomain A (ED-A), ED-B, and connecting segment (CS) III. A middle region of FN containing homologous repeating segments of about 90 amino acids, called type III homologies, has been identified.^24,25 Using immunologic techniques with monoclonal antibodies, it was shown that the ED-A exon is omitted during splicing of the FN mRNA precursor in arterial medial cells; the expression of FN containing ED-A, however, is characteristic of multiple cell types involved in wound healing and tissue and organ fibrotic diseases characterized by the overproduction of connective tissue proteins. In such disorders, EDA-FN synthesis precedes that of collagens and is a requirement for the TGF-β–induced differentiation of fibroblasts into myofibroblasts. The contributions of myofibroblast differentiation and expression of the EDA-FN isoform to the aging process are problematic because of their close association with the early stages of fibrotic diseases.²⁶ Genetic studies have shown that ED-A is not required for normal development, but significant abnormalities were noted in adult mice that lacked the ED-A gene.²⁷ Increased ED-A FN has been demonstrated in the skin of patients with scleroderma.²⁸ ED-A FN also is found during embryonic development where it plays a role in cell migration. In addition, recent evidence has shown the presence of EDA-FN in keloid scars.²⁹

This could be the source of differences between the plasma and cellular forms of FN. This phenomenon of alternative splicing may also be involved in the synthesis of collagens and elastin and may well be implicated in the processes of aging.

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