ABSTRACT

The great preponderance of evidence that has accrued over the past three decades suggests that proteoglycans (PGs), a relatively small subset of glycoproteins, may be among the most useful surfacederivatizing compounds available to biomedical science. The defin-ing characteristic of these specialized glycoproteins is the presence of variable numbers of linear, O-linked acidic polysaccharide sugar chains known as glycosaminoglycans (GAGs). PGs reside on the plasma membrane of almost all animal cells studied so far and so constitute a major component of the extracellular matrix (ECM). All proteins outside the cell, regardless of their role, have evolved in the presence of these highly sulfated polysaccharides since the advent of metazoa. Studies of model organisms and human diseases have demonstrated the key roles of these complexes during development, normal cellular physiology, and wound healing as they are known

to participate in the fine-tuning of several key processes, including blood coagulation, tissue assembly, adhesion, motility, virus trans-duction, cell growth, and morphogenesis. Of interest here is the electrostatic interaction of GAG chains, particularly heparan sulfate (HS) chains, with protein ligands. The central importance of these interactions is borne out by phenotypic studies of mice and humans bearing crippling mutations in either the core proteins or the bio-synthetic enzymes responsible for assembling GAG chains. Seminal studies in the 1990s that established that HS played a central role in controlling the bioactivities, not just of large adhesive molecules, such as laminin and fibronectin, but also of heparin-binding growth factors, most particularly fibroblast growth factors (FGFs), forever changed the way we think of the functional role of GAG sugars. A motivating principle for a new generation of GAG-coated biomaterials has thus become not to coat implant surfaces with fragile and expensive proteins or peptides but rather with the GAG sugars that normally trigger the bioactivities of such proteins, so better and more efficiently triggering natural integration mechanisms. Such an approach offers enormous opportunities not only for cellular studies but also for new generations of tailored bioscaffolds, as GAGs, unlike proteins, are chemically robust and withstand rigorous industrial processing. 17.1 Proteoglycans: Core Proteins and GAG

Sugars (Mulloy)The basic PG structure consists of a protein core to which one or more linear GAG chains are covalently attached. PGs are categorized depending upon the nature of their GAG chains, either chondroitin/dermatan sulfate (CS/DS), HS, or keratan sulfate (KS) PGs, or by their size (where versican or aggrecan are classed as large, and the leucine-rich repeat PGs such as decorin or biglycanare classed as small). The GAG chains are linked to a serine residue in the core protein by a tetrasaccharide linker. Heparin, in contrast, is cleaved from its core protein, serglycin, and is secreted from mast cells as a free GAG chain. Virtually all mammalian cells express HSPGs, and depending on the core protein, they may either become associated with the cell surface or deposited into the ECM[1]. The linear polysaccharide backbone of GAGs consists of a repeat-ing disaccharide comprising an N-acetyl hexosamine alternating

with β-D-glucuronic acid(GlcA), α-L-iduronic acid (IdoA), or galac-tose. There are four classes of GAG, each distinguished by a particu-lar repeating disaccharide.Of the four classes of GAG, hyaluronic acid is unique in that once the chain is elongated, it is not subjected to any further sulfation or modification. DS is essentially a CS where the D-glucuronic acid is extensively epimerized to give L-iduronic acid. KS and CS, however, are modified, although less exuberantly than HS/heparin. The HS/heparin class is based on a repeat disaccharide backbone of glucuronic acid-(β1-4)-N-acetylglucosamine-(α1-4) and undergoes the largest number of modifications, particularly sul-fations, and so has a greater sequence variability and higher overall negative charge density compared with other GAG classes [2]. This chain complexity arises during its biosynthesis in the Golgi, for as even as the GAG chain is being elongated, the incorporated disaccharides are subject to a further set of intricate modifications, in-cluding partial deacetylation, epimerization on the glucuronic acid, and substitution with N-and O-sulfates to result in highly complex, heterogeneous structures [3]. Although binding interactions of the HS/heparin GAGs with peptide growth factors and other proteins have been the subject of intense study, the potential importance of CS/DS sequences has also become apparent. Employing a combination of synthetic chemistry, microarray technology, and biological assays, Gama et al. [4] demonstrated a set of CS tetrasaccharides whose interactions and bioactivity provides evidence for a “sulfationcode” in which recognition of selected proteins is conferred by specific sequences [5]. This is clinically important because during the latter phases of osteogenesis, bone ECM undergoes a change in its predominant PG species from HSPGs to CSPGs. The latter, although now thought to have growth-promoting activities, are generally less able to potentiate growth factor activity, owing to their relative structural simplicity, and have been posited to be secreted, at least in part, to create stable adult tissue forms as they bind calcium, mineralize, and maintain tissue hydration. The growth-promoting effects of the HSPGs thus give way to the increasing need for the structural integrity that is supported by the CSPGs [6]. Further discussion will therefore focus on the heparin/HS class of GAGs, as they currently appear to be the most useful for the derivatization of surfaces.