EXTRACELLULAR MATRIX | Matrix Proteoglycans

184 EXTRACELLULAR MATRIX / Matrix Proteoglycans

Matrix Proteoglycans C W Frevert, VA Puget Sound Medical Center, Seattle, WA, USA T N Wight, The Hope Heart Institute, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Matrix proteoglycans, complex molecules composed of a core protein and glycosaminoglycan side chains, impart biomechanical properties to lung tissue and are important biological modifiers which regulate processes such as lung development, homeostasis, inflammation, and wound healing. The diverse roles of matrix proteoglycans suggest that these molecules play a critical role in normal and diseased lungs.

are four classes of glycosaminoglycans: (1) hyaluronan, (2) chondroitin sulfate (CS)/dermatan sulfate (DS), (3) heparan sulfate (HS)/heparin, and (4) keratan sulfate (KS). All four classes of glycosaminoglycan are found in normal lungs and all except hyaluronan are bound to core proteins. The predominant glycosaminoglycan in normal lungs is HS (40–60%) followed by CS/DS (31%), hyaluronan (14%), and heparin (5%). Sulfation of glycosaminoglycans, a structural modification that occurs in the Golgi during chain elongation, has important biological consequences as specific sulfation patterns on glycosaminoglycans form the binding sites for a number of proteins including morphogens, growth factors, cytokines, and chemokines.

Introduction

Glycosaminoglycan Structure

Proteoglycans are a family of charged molecules containing a core protein and one or more covalently attached glycosaminoglycan side chains. The complexity and diversity of proteoglycans is derived from approximately 30 different core proteins of varying size and structure (10–500 kDa) and the considerable variability in the number (1 to 4100) and types of glycosaminoglycan side chains attached. Proteoglycans are found in the extracellular matrix, plasma membrane of cells, and intracellular structures. Matrix proteoglycans such as perlecan, collagen XVIII, and agrin are found in the basal laminal of cells, and decorin, biglycan, and versican are found in the interstitial spaces of the lungs. The glycosaminoglycan side chains and core proteins of matrix proteoglycans regulate a number of biomechanical and cellular processes in normal and diseased lungs.

Hyaluronan is a repeating disaccharide structure composed of glucuronic acid and N-acetyl glucosamine that is nonsulfated and is not attached to a core protein (Table 1). CS has a disaccharide repeat pattern similar to hyaluronan, but it contains galactosamine instead of glucosamine. The galactosamine can have a sulfate ester attached at the 4- or 6-position (e.g., chondroitin 4-sulfate) and the glucuronic acid can have a sulfate ester placed at the 2-position. The degree of sulfation of CS is variable and can differ within a single preparation and from one tissue to another. Dermatan sulfate is a copolymer composed of two types of disaccharide repeats, D-glucuronate-N-acetyl galactosamine and L-iduronate-N-acetyl galactosamine. The formation of iduronic acid residues occurs by the conversion of glucuronic acid already incorporated into the growing polymer by an epimerization reaction that is tightly coupled to the sulfation process. KS has a repeating disaccharide residue containing galactose and N-acetyl glucosamine. The linkage of KS to the core protein differs from other glycosaminoglycans because it is not formed on the typical xylose–serine linkage. KS expression in lungs is

Glycosaminoglycan Side Chains Glycosaminoglycans are linear polymers of repeating disaccharides with a high negative charge imparted by sulfate and/or carboxyl groups in their structure. There

Table 1 Structure of glycosaminoglycans Glycosaminoglycan

Monosaccharide (1)

Monosaccharide (2)

Sulfation patterns

Hyaluronan Chondroitin sulfate

D-glucuronic acid D-glucuronic acid

N-acetyl glucosamine N-acetyl galactosamine

Dermatan sulfate

D-glucuronic acid or L-iduronic acid D-galactose D-glucuronic acid or L-iduronic acid D-glucuronic acid or L-iduronic acid

N-acetyl galactosamine

No sulfation 2-O-sulfation, 4-O-sulfation, 6-O-sulfation 2-O-sulfation, 4-O-sulfation, 6-O-sulfation 6-O-sulfation 2-O-sulfation, 3-O-sulfation, 6-O-sulfation, N-sulfation 2-O-sulfation, 3-O-sulfation, 6-O-sulfation, N-sulfation

Keratan sulfate Heparan sulfate Heparin

N-acetyl glucosamine N-acetyl glucosamine N-acetyl glucosamine

EXTRACELLULAR MATRIX / Matrix Proteoglycans

limited but this glycosaminoglycan is present in the cartilage of the trachea. Heparin and HS are glycosaminoglycans of closely related structure and contain repeating disaccharide units composed of glucosamine and either D-glucuronic acid or L-iduronic acid. Glucuronic acid is the predominant uronic acid component in HS, whereas iduronic acid is the largest component in heparin. Heparin and HS contain a number of glucosamine residues that contain N-sulfate groups instead of N-acetyl groups. HS contains a lower degree of O-sulfation than heparin and therefore has fewer sulfates per disaccharide than heparin. In mature HS about 40–50% of the amino sugars are converted from the N-acetylated form to the N-sulfated form. The N-sulfated residues occur predominantly in contiguous sequences or S-domains, which are separated by 15 disaccharides that are made in large part by N-acetyl-rich domains. This results in HS being a more complex molecule than heparin.

Core Proteins of Matrix Proteoglycans A number of HS proteoglycans (HSPGs), CS proteoglycans (CSPGs), and DS proteoglycans (DSPGs) are found in the matrix of lungs (Table 2). Perlecan is a large proteoglycan consisting of five domains, which contain low-density lipid (LDL) receptor class A modules, Ig-like repeats, laminin-like repeats, and EGF-like repeats. Electron micrographs of perlecan showed a protein with the appearance of beads on a string, which resulted in the name perlecan. Whereas HS is the predominant glycosaminoglycan on perlecan, CS chains are found on domain V of recombinant perlecan. Collagen XVIII is an HSPG with features typical of collagen including sensitivity of the core protein to collagenase. This HSPG is of particular interest because of the presence of endostatin, a 22 kDa antiangiogenic peptide located in the C-terminal domain of collagen XVIII. Agrin is an HSPG that contains nine follistatin-like domains which share similarity to Kazal-type protease inhibitors, including pancreatic trypsin inhibitor, follistatin, thrombin inhibitor, and elastase inhibitor. The N-terminal laminin-binding domain of chick agrin has a high

Table 2 Matrix proteoglycans Proteoglycan

Core protein (kDa)

Location

GAG type

Perlecan Collagen XVIII Agrin Decorin Biglycan Versican

450 180 220 40 40 350

Basal lamina Basal lamina Basal lamina Interstitium Interstitium Interstitium

HS/CS HS HS/CS CS/DS CS/DS CS

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structural similarity to the protease inhibition domain in tissue inhibitor of metalloproteinase-1. Decorin and biglycan are small leucine-rich CSPGs with core proteins of similar size and structure (Table 2). The core protein of decorin has one CS/DS chain, whereas biglycan has two CS/DS chains. Versican is a large CSPG with a core protein consisting of an N-terminal globular domain (G1) which binds hyaluronan and a C-terminal binding domain (G3) which contains lectin-like, EGF-like, and CRP-like subdomains. The two globular domains, G1 and G3, are separated by two domains that contain CS chains. Alternative splicing of versican mRNA leads to four isoforms, V0, V1, V2, and V3, which carry varying amounts of CS side chains, and mRNA for all isoforms except V2 have been found in normal lungs.

Distribution of Matrix Proteoglycans Basement membrane proteoglycans. The basement membrane or basal lamina of the cell contains proteoglycans that interact directly with cells (Figure 1). Proteoglycans found in the basement membrane include the HSPGs, perlecan, agrin, and collagen XVIII. Collagen IV is the only collagen which is more abundant in the basement membrane than collagen XVIII. Interstitial proteoglycans. The large CS proteoglycan, versican, is located in the interstitial spaces in the lungs where it interacts with hyaluronan (HA), which fixes this proteoglycan in tissue as a very high-molecular-weight aggregate (Figure 1). Decorin and biglycan are also located in the interstitial spaces of the lungs where decorin binds collagen fibers with a regular periodicity. Basal surface of cells. Syndecans are a small family of transmembrane HSPGs which interact with a number of matrix proteins (Figure 1). Whereas syndecans are not considered matrix proteoglycans (and are therefore not discussed in detail in this article), their interaction with matrix transmits signals plays a role in focal adhesion and cell death pathways. Syndecans are described in greater detail in Extracellular Matrix: Surface Proteoglycans.

Biomechanical Properties of Proteoglycans Extracellular matrix proteoglycans confer important biomechanical properties to lung tissue. Glycosaminoglycans are strongly hydrophilic and their high negative charge attracts cations such as Naþ , which pulls water into the matrix through osmotic activity. The large amount of water pulled into the

186 EXTRACELLULAR MATRIX / Matrix Proteoglycans

Alveolar space

Type II cell

Alveolar macrophage

Epithelium

Interstitial space

Perlecan Syndecan-1

Collagen

Versican-HA Interstitial cell

Decorin Syndecans

Endothelium

Vascular space Figure 1 Matrix proteoglycans in normal lungs. Perlecan, a HS proteoglycan, is found in the basal lamina of epithelial and endothelial cells. The CS proteoglycans, versican and decorin, are found in the interstitial space of the lungs. Versican binds to the glycosaminoglycan, hyaluronan, to form high-molecular-weight complexes. Decorin binds to collagen and helps stabilize the collagen–elastin network. Syndecans are membrane proteoglycans that interact with matrix proteins. This figure is not meant to represent the concentrations of the different proteoglycans in normal lungs and is meant to only show their location in lung tissue.

matrix by glycosaminoglycans results in swelling of the matrix, which allows the matrix to withstand compressive forces. Proteoglycans interact with other matrix molecules such as collagen, laminin, and elastin to form a structured matrix. Work performed in decorin knockout mice suggests that the stabilization of the collagen–elastin network by this proteoglycan contributes to lung elasticity and alveolar stability. Proteoglycans also provide a selective charge filtration barrier for the exchange of oxygen and plasma constituents and studies suggest that disruption of this charge barrier could lead to marked alterations in vascular permeability.

Proteoglycans as Determinants of Cellular Phenotype Proteoglycans are important determinants of cellular phenotype, playing a critical role in development, cellular proliferation, cell death, inflammation, and wound healing. A critical function of proteoglycans

in matrix is mediated by glycosaminoglycan side chains which bind to growth factors, cytokines, and chemokines. The binding of these proteins to glycosaminoglycans was initially believed to be a non-specific ionic interaction between positively charged amino acids on proteins and the negatively charged sulfates on glycosaminoglycans. It is now known that considerable specificity exists in the interaction between proteins and glycosaminoglycans with unique binding sites being identified on glycosaminoglycans for proteins such as thrombin and growth factors. The degree of specificity between proteins and glycosaminoglycans is still not known but there is speculation that there may be a specific sulfation pattern (e.g., binding site) for each protein that binds to a glycosaminoglycan. Current evidence suggests that the specificity observed in protein–glycosaminoglycan interactions plays an important role in the function of proteins in lung tissue. Listed below are five potential functions of protein– glycosaminoglycan interactions.

EXTRACELLULAR MATRIX / Matrix Proteoglycans

1. Determination of protein binding sites. Because of the specificity observed in protein–glycosaminoglycan interactions, matrix proteoglycans position proteins such as chemokines and fibroblast growth factors to distinct anatomical locations (e.g., basement membrane or intersitial spaces). 2. Storage/sequestration. Glycosaminoglycans provide a site for proteins to bind, which sequesters a protein and prolongs its retention in lung tissue. The binding of chemokines to glycosaminoglycans provides a site for dimerization of chemokines which is a mechanism shown to increase the amount of chemokine binding in the lungs. Undersulfation of the basement membrane matrix of alveolar type II cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these alveolar epithelial cells. Increased sulfation of glycosaminoglycans under type I cells sequesters fibroblast growth factors, limiting their effect on this cell. In contrast, decreased sulfation of the matrix under type II cells allows fibroblast growth factors to activate signaling pathways in this cell. 3. Formation and stabilization of morphogen and chemokine gradients. The binding of morphogens and chemokines to glycosaminoglycans has been proposed as a mechanism whereby gradients develop in tissue. The binding affinity of chemokines to glycosaminoglycans, which is in the low micromolar range, provides a mechanism whereby the chemokine is able to diffuse and form chemotactic gradients in tissue. 4. Cell signaling. The binding of growth factors and cytokines to glycosaminoglycans has been shown to play a role in cellular activation. For proper presentation to fibroblast growth factor receptors, members of the fibroblast growth factor family interact with HSPG. 5. Protection from proteolysis. The binding of a protein to a glycosaminoglycan may sequester proteolytic cleavage sites, which protects proteins from proteolysis. Glycosaminoglycans have been shown to protect chemokines and growth factors from proteolytic cleavage.

Proteoglycans and Disease Changes in the composition of glycosaminoglycans and proteoglycans in the lungs have been reported in animal models and human lung disease. A consistent finding in animal models such as exposure to lipopolysaccharide, bleomycin, and silica is an increase in the synthesis of CS and DS. This change occurs within the first 72 h following exposure and results in

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a shift from the normal lung with HS predominating to the diseased lung where the predominant GAG is CS and DS. An increased deposition of the CSPG, versican, is a feature common to a number of interstitial lung diseases, including acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis, bronchiolitis obliterans organizing pneumonia, and lymphangioleiomyomatosis. Often associated with the increased expression of versican in the lungs is an increased deposition of hyaluronan and the CSPGs, decorin and biglycan. Patients with severe asthma have increased expression of versican and biglycan in their airways, which is correlated with airway responsiveness. Because of the potential for matrix PG and hyaluronan to affect mechanical properties and cellular function in the lungs, changes in their expression will most likely affect the clinical course of lung diseases. However, further work is required to determine the role of proteoglycans in the pathogenesis of lung disease. See also: Alveolar Wall Micromechanics. Extracellular Matrix: Matricellular Proteins; Surface Proteoglycans. Fibroblast Growth Factors. Smooth Muscle Cells: Airway.

Further Reading Allen BL, Filla MS, and Rapraeger AC (2001) Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition. Journal of Cell Biology 155(5): 845–858. Bensadoun ES, Burke AK, Hogg JC, and Roberts CR (1996) Proteoglycan deposition in pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 154(6 Pt 1): 1819–1828. Cavalcante FS, Ito S, Brewer K, et al. (2005) Mechanical interactions between collagen and proteoglycans: implications for the stability of lung tissue. Journal of Applied Physiology 98(2): 672–679. Frevert CW, Kinsella MG, Vathanaprida C, et al. (2003) Binding of interleukin-8 to heparan sulfate and chondroitin sulfate in lung tissue. American Journal Respiratory Cell and Molecular Biology 28(4): 464–472. Fust A, LeBellego F, Iozzo RV, Roughley PJ, and Ludwig MS (2005) Alterations in lung mechanics in decorin-deficient mice. American Journal of Physiology: Lung Cellular and Molecular Physiology 288(1): L159–L166. Giri SN, Hyde DM, Braun RK, et al. (1997) Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis. Biochemical Pharmacology 54(11): 1205–1216. Halfter W, Dong S, Schurer B, and Cole GJ (1998) Collagen XVIII is a basement membrane heparan sulfate proteoglycan. Journal of Biological Chemistry 273(39): 25404–25412. Karlinsky JB, Bucay PJ, Ciccolella DE, and Crowley MP (1991) Effects of intratracheal endotoxin administration on hamster lung glycosaminoglycans. American Journal of Physiology 261(2 Pt 1): L148–L155. Noonan DM, Fulle A, Valente P, et al. (1991) The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. Journal of Biological Chemistry 266(34): 22939–22947.

188 EXTRACELLULAR MATRIX / Surface Proteoglycans Sannes PL, Khosla J, Li CM, and Pagan I (1998) Sulfation of extracellar matrices modifies growth factor effects on type II cells on laminin substrata. American Journal of Physiology 275(4 Pt 1): L701–L708. Sasaki T, Fukai N, Mann K, et al. (1998) Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO Journal 17(15): 4249–4256. Smits NC, Robbesom AA, Versteeg EM, et al. (2004) Heterogeneity of heparan sulfates in human lung. American Journal of respiratory Cell and Molecular Biology 30(2): 166–173. Spillmann D, Witt D, and Lindahl U (1998) Defining the interleukin-8-binding domain of heparan sulfate. Journal of Biological Chemistry 273(25): 15487–15493. Tapanadechopone P, Hassell JR, Rigatti B, and Couchman JR (1999) Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan. Biochemical and Biophysical Research Communications 265(3): 680–690. Westergren-Thorsson G, Chakir J, Lafreniere-Allard MJ, Boulet LP, and Tremblay GM (2002) Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects. International Journal of Biochemistry & Cell Biology 34(10): 1256–1267.

Surface Proteoglycans P W Park, Y Chen, and K Hayashida, Baylor College of Medicine, Houston, TX, USA

defines proteoglycans as heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), or keratan sulfate proteoglycans (KSPGs). Some proteoglycan core proteins carry both heparan sulfate (HS) and chondroitin sulfate (CS) chains. Surface proteoglycans include the syndecan and glypican families with four and six members in mammals respectively, NG2 (AN2 in mice), betaglycan, thrombomodulin, a5b1 integrin, and CD44. The first studies on GAGs and proteoglycans date back to 1916 when heparin, a highly sulfated version of HS, was unexpectedly identified as a potent anticoagulant in liver extracts (hence the name heparin) by a medical student who was trying to isolate a procoagulant molecule. HS was first thought to be a contaminant in the heparin preparation, but was later distinguished from heparin in 1948 by the difference in the extent of sulfation and greater structural variability. For a long time, biological functions of proteoglycans were largely speculative and, in fact, most proteoglycans were thought to be specific to cartilage, functioning as cushions in joints for variable, compressive loads. Recent studies have revealed that surface proteoglycans also function as key modulators of many molecular interactions, including those relevant to lung biology.

& 2006 Elsevier Ltd. All rights reserved.

Structure Abstract Cell surface proteoglycans, such as syndecans, glypicans, and CD44, regulate a wide variety of molecular interactions that mediate cell adhesion, migration, proliferation, and differentiation. Through these activities, surface proteoglycans modulate many pathophysiological processes, including development, inflammation, tissue repair, cancer metastasis, and infection. Proteoglycans are composites of glycosaminoglycans (GAGs) attached covalently to core proteins. The majority of the ligandbinding activities of proteoglycans are mediated through GAGs. Recent studies have revealed that surface proteoglycans play key roles in lung biology. In humans, expression of several surface proteoglycans is reduced in lung cancers, suggesting that these proteoglycans are potential prognostic markers. Mice lacking certain GAG biosynthetic enzymes die soon after birth from conditions resembling acute respiratory distress syndrome (ARDS), indicating that proteoglycans are essential for lung development and function. Furthermore, inflammatory responses in mice made null for syndecan-1 and CD44 are drastically altered in various models of lung inflammation. These data highlight the physiological significance of surface proteoglycans in lung development and in the pathogenesis of respiratory diseases.

Introduction Nearly all mammalian cells express proteoglycans on their cell surface. The chemical nature of the glucosaminoglycans (GAGs) attached to core proteins

A proteoglycan consists of a core protein and one or several covalently attached GAG chains. GAGs are attached to and polymerized on certain Ser residues of a Ser-Gly dipeptide sequence, often repeated two or more times. GAGs are linear polysaccharides consisting of repeating disaccharide units that are defined by the composition and chemical linkage of the amino sugar and uronic acid monosaccharides in the disaccharide unit. The signature disaccharide repeat of an HS/heparin polysaccharide is GlcUAb1-4GlcNAca1–4, CS (including dermatan sulfate) is GlcUAb1–3GalNAcb1–4, keratan sulfate (KS) is Galb1-4GalNAcb1–3, and hyaluronan (HA) is GlcUAb1–3GlcNAcb1–4. Except for HA, these primary structures are modified in the Golgi apparatus by several sulfation and epimerization reactions that are catalyzed by distinct enzymes. Because the polymerization and modification reactions do not go to completion, the biosynthetic process generates an exceptionally diverse array of GAG structures, both in length and extent of modification. For example, HS, the most structurally heterogeneous GAG, varies in length from 50 to 150 disaccharides with cell type and core protein. However, a mere HS decasaccharide can potentially assume over 106 distinct sequences, which is already in vast excess of the estimated gene products