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GAGs are classified into several major types based on the nature of the repeating disaccharide unit: heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. A hypersulfated form of heparan sulfate called heparin, produced mostly by mast cells, plays a key role in allergic reactions. It is also used medically as an anticlotting drug because of its ability to activate a natural clotting inhibitor called antithrombin III.

As we will see in later chapters, complex signaling pathways direct the emergence of various cell types in the proper position and at the proper time in normal embryonic development. Laboratory generation and analysis of mutants with defects in proteoglycan production in Drosophila melanogaster (fruit fly), C. elegans (roundworm), and mice have clearly shown that proteoglycans play critical roles in development, most likely as modulators of various signaling pathways.

Biosynthesis of Proteoglycans With the exception of hyaluronan, which is discussed in the next section, all the major GAGs occur naturally as components of proteogly-cans. Like other secreted and transmembrane glycoproteins, proteoglycan core proteins are synthesized on the endoplas-mic reticulum (Chapter 16). The GAG chains are assembled on these cores in the Golgi complex. To generate heparan or chondroitin sulfate chains, a three-sugar "linker" is first attached to the hydroxyl side chains of certain serine residues in a core protein (Figure 6-18). In contrast, the linkers for the addition of keratan sulfate chains are oligosaccharide chains attached to asparagine residues; such A-linked oligosaccha-rides are present in most glycoproteins, although only a subset carry GAG chains. All GAG chains are elongated by the alternating addition of sugar monomers to form the disac-charide repeats characteristic of a particular GAG; the chains are often modified subsequently by the covalent linkage of small molecules such as sulfate. The mechanisms responsible for determining which proteins are modified with GAGs, the sequence of disaccharides to be added, the sites to be sul-

(GlcUA—GalNAc)n—GlcUA—Gal—Gal — Xyl—Ser

- Core protein

Chondroitin sulfate repeats

Linking sugars

Gal = galactose

GalNAc = N-acetylgalactosamine

GlcUA = glucuronic acid Xyl = xylose

▲ FIGURE 6-18 Biosynthesis of heparan and chondroitin sulfate chains in proteoglycans. Synthesis of a chondroitin sulfate chain (shown here) is initiated by transfer of a xylose residue to a serine residue in the core protein, most likely in the Golgi complex, followed by sequential addition of two galactose residues. Glucuronic acid and ^-acetylgalactosamine residues are then added sequentially to these linking sugars, forming the chondroitin sulfate chain. Heparan sulfate chains are connected to core proteins by the same three-sugar linker.

fated, and the lengths of the GAG chains are unknown. The ratio of polysaccharide to protein in all proteoglycans is much higher than that in most other glycoproteins.

Diversity of Proteoglycans The proteoglycans constitute a remarkably diverse group of molecules that are abundant in the extracellular matrix of all animal tissues and are also expressed on the cell surface. For example, of the five major classes of heparan sulfate proteoglycans, three are located in the extracellular matrix (perlecan, agrin, and type XVIII collagen) and two are cell-surface proteins. The latter include integral membrane proteins (syndecans) and GPI-anchored proteins (glypicans); the GAG chains in both types of cell-surface proteoglycans extend into the extracellular space. The sequences and lengths of proteoglycan core proteins vary considerably, and the number of attached GAG chains ranges from just a few to more than 100. Moreover, a core protein is often linked to two different types of GAG chains (e.g., heparan sulfate and chondroitin sulfate), generating a "hybrid" proteoglycan. Thus, the molecular weight and charge density of a population of proteoglycans can be expressed only as an average; the composition and sequence of individual molecules can differ considerably.

Perlecan, the major secreted proteoglycan in basal laminae, consists of a large multidomain core protein (=400 kDa) with three or four specialized GAG chains. 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 of its multiple domains with distinctive binding properties, perlecan can cross-link not only ECM components to one another but also certain cell-surface molecules to ECM components.

Syndecans are expressed by epithelial cells and many other cell types. These cell-surface proteoglycans bind to collagens and multiadhesive matrix proteins such as the fibronectins, which are discussed in Section 6.4. In this way, cell-surface proteoglycans can anchor cells to the extracellular matrix. Like that of many integral membrane proteins, the cytosolic domain of syndecan interacts with the actin cytoskeleton and in some cases with intracellular regulatory molecules. In addition, cell-surface proteoglycans bind many protein growth factors and other external signaling molecules, thereby helping to regulate cellular metabolism and function. For instance, syndecans in the hypothalamic region of the brain modulate feeding behavior in response to food deprivation (fasted state). They do so by participating in the binding of antisatiety pep-tides to cell-surface receptors that help control feeding behavior. In the fed state, the syndecan extracellular domain decorated with heparan sulfate chains is released from the surface by proteolysis, thus suppressing the activity of the anti-satiety peptides and feeding behavior. In mice engineered to overexpress the syndecan-1 gene in the hypothalamic region of the brain and other tissues, normal control of feeding by anti-satiety peptides is disrupted and the animals overeat and become obese. Other examples of proteoglycans interacting with external signaling molecules are described in Chapter 14.

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M FIGURE 6-19 Pentasaccharide GAG sequence that regulates the activity of antithrombin III (ATIII).

Sets of modified five-residue sequences in heparin with the composition shown here bind to ATIII and activate it, thereby inhibiting blood clotting. The sulfate groups in red type are essential for this heparin function; the modifications in blue type may be present but are not essential. Other sets of modified GAG sequences are thought to regulate the activity of other target proteins. [Courtesy of Robert Rosenberg and Balagurunathan Kuberan.]

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Modifications in Glycosaminoglycan (GAG) Chains Can Determine Proteoglycan Functions

As is the case with the sequence of amino acids in proteins, the arrangement of the sugar residues in GAG chains and the modification of specific sugars (e.g., addition of sulfate) in the chains can determine their function and that of the pro-teoglycans containing them. For example, groupings of certain modified sugars in the GAG chains of heparin sulfate proteoglycans can control the binding of growth factors to certain receptors, the activities of proteins in the blood-clotting cascade, and the activity of lipoprotein lipase, a membrane-associated enzyme that hydrolyzes triglycerides to fatty acids (Chapter 18).

For years, the chemical and structural complexity of proteoglycans posed a daunting barrier to an analysis of their structures and an understanding of their many diverse functions. In recent years, investigators employing classical and new state-of-the-art biochemical techniques (e.g., capillary high-pressure liquid chromatography), mass spec-trometry, and genetics have begun to elucidate the detailed structures and functions of these ubiquitous ECM molecules. The results of ongoing studies suggest that sets of sugar-residue sequences containing some modifications in common, rather than single unique sequences, are responsible for specifying distinct GAG functions. A case in point is a set of five-residue (pentasaccharide) sequences found in a subset of heparin GAGs that control the activity of antithrombin III (ATIII), an inhibitor of the key blood-clotting protease thrombin. When these pentasaccharide sequences in heparin are sulfated at two specific positions, heparin can activate ATIII, thereby inhibiting clot formation (Figure 6-19). Several other sulfates can be present in the active pentasaccharide in various combinations, but they are not essential for the anticlotting activity of he-parin. The rationale for generating sets of similar active sequences rather than a single unique sequence and the mechanisms that control GAG biosynthetic pathways, permitting the generation of such active sequences, are not well understood.

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