Emerging Views of Heparan Sulfate Glycosaminoglycan Structure

Home , Heparan sulfate

chain contains a ␤-D-glucosamine linked to an uronic acid, which can be one of two C5 epimers, either ␣-L-iduronic or ␤-D-glucuronic acid. Other than the C5 Emerging Views of Heparan Sulfate position of the uronic acid, HLGAGs Glycosaminoglycan Structure/Activity vary in their chain length, that is, the number of disaccharide repeat units, and Relationships Modulating Dynamic degree of sulfation and acetylation of each disaccharide unit. O-sulfation of the Biological Functions disaccharide repeat can occur at the 2-O Zachary Shriver, Dongfang Liu, and Ram Sasisekharan* position of the uronic acid and the 6-O and 3-O positions of the glucosamine. Thus, for a given disaccharide unit within an HSGAG structure, each site is Heparan sulfate glycosaminoglycans (HSGAGs) are an important subset either sulfated or unsubstituted, creat- of complex polysaccharides that represent the third major class of biopoly- ing eight possible combinations. In ad- mer, along with polynucleic acids and polypeptides. However, the impor- dition, as mentioned above, there are two possibilities for the uronic acid com- tance of HSGAGs in biological processes is underappreciated because of a ponent of each disaccharide unit, that lack of effective molecular tools to correlate specific structures with func- is, either iduronic acid or glucuronic tions. Only recently have significant strides been made in understanding acid, giving rise to a total of 16 different the steps of HSGAG biosynthesis that lead to the formation of unique possible disaccharide combinations. Fi- nally, the N-position of the glucosamine structures of functional importance. Such advances now create possibili- can be sulfated, acetylated, or unsubsti- ties for intervening in numerous clinical situations, creating much-needed tuted (three possible states). Taking this novel therapies for a variety of pathophysiological conditions including final factor into account results in 48 atherosclerosis, thromboembolic disorders, and unstable angina. (Trends possible disaccharide building blocks. Cardiovasc Med 2002;12:71–77). © 2002, Elsevier Science Inc. Thus, HSGAGs have the potential to carry more information than any other biopolymer, including either DNA (made up of four bases) or proteins (made up Heparan sulfate-like glycosaminoglycans to be important regulators of biological of 20 amino acids). Consider, for in- (HSGAGs) are complex acidic polysac- processes ranging from embryogenesis stance, a simple polymer of each kind charides present both within the extra- (Perrimon and Bernfield 2000) to hemo- made of six units. For DNA, there are 46 cellular matrix (ECM) that surrounds stasis (Rosenberg et al. 1997) as well as or 4096 possible sequences for this hex- cells and at the cell surface as protein– in the pathophysiology of disease states, amer. In contrast, for a hexapeptide, carbohydrate conjugates (termed proteo- such as atherosclerosis and thromboem- there are many more possibilities, 206 or glycans) (Figure 1). HSGAG proteogly- bolic disorders (Kolset and Salmivirta 64 million possible permutations. How- cans are found at the surface of virtually 1999). This review focuses on recent sci- ever, for HSGAGs, a polymer made of every cell type, where they act as impor- entific advances that have furthered our six disaccharide units could have a total tant biological mediators of various cell- understanding of how HSGAG structure of over 12 billion possible sequences, a related events such as proliferation, impinges on function. Specific emphasis staggering degree of variation, over 100 morphogenesis, adhesion, migration, and will be placed upon how these advances times as much as for polypetides and cell death (apoptosis) (Sasisekharan have increased our appreciation of the two million times that of DNA! and Venkataraman 2000; Tumova et al. importance of complex polysaccharides 2000). Given their role in fundamental in vascular biology, and how this under- • HSGAGs in Cardiovascular cellular events, HSGAGs have been found standing offers new potential for the de- Hemostasis velopment of novel classes of therapeutics. That HSGAGs can regulate such a va- Given the information density of HSGAGs, Zachary Shriver, Dongfang Liu, and Ram riety of biological processes is a function each HSGAG chain contains multiple Sasisekharan are from the Division of Bioengineering and Environmental Health of the chemical diversity inherent in the sequences that specifically regulate mul- and the Center for Biomedical Engineering, HSGAG polymer. A disaccharide repeat tiple components of the cardiovascular Massachusetts Institute of Technology, Cam- unit characterizes all HSGAGs; however, system (Figure 2a). In some cases, it has bridge, MA. HSGAGs from various sources differ been shown that certain HSGAG struc- * Address correspondence to: Dr. R. Sasise- both in terms of their length (that is, the tures provide protective effects against kharan, MIT, 77 Massachusetts Avenue, number of disaccharide repeat units) and disease processes such as atherosclero- Building 16-561, Cambridge, MA 02139, sis and unstable angina (Kanabrocki et USA. Tel.: (ϩ1) 617-258-9494; fax: (ϩ1) 617- differential chemical modifications within 258-9409; e-mail: [email protected]. the HSGAG chain (Figure 1) (Salmivirta al. 1992). For instance, HSGAGs lining © 2002, Elsevier Science Inc. All rights et al. 1996; Turnbull et al. 2001). the luminal surface of endothelial cells reserved. 1050-1738/02/$-see front matter Each disaccharide unit of an HSGAG effectively provide electronegativity, and

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the atherogenic process. The pharmacological administration of highly sulfated HSGAGs restores the electronegativity of the damaged endothelium lining, thereby limiting further damage and preventing thrombosis. In addition, cell surface HSGAGs, through interactions with lipoprotein lipase and apolipoproteins B and E, are involved in the metabolism of lipoproteins including HDL by regulating their uptake and metabolism (Figure 2a) (Kolset and Salmivirta 1999, Rosenberg et al. 1997). In this manner, HSGAGs mediate the “lipid clearing effect” of plasma. Finally, cell surface HSGAGs, depending on structure and concentration, can either inhibit or promote growth factor signaling, thus regulating SMC proliferation during vascular stenosis and atherosclerosis. Importantly, many of these biological observations have been successfully translated to the clinic; specific examples will be illustrated below. The protective effects of highly sulfated HSGAGs have been extensively demonstrated in coronary diseases such as myo- cardial infarction and unstable angina. Thus, HSGAG structures impinge on vascular biology in multiple ways. To differentiate roles for specific HSGAG sequences requires the application of newly developed technologies for the isolation and characterization of biologically important HSGAG sequences. Be- low, we focus on two specific examples where such studies have offered a clear understanding of how specific HSGAG structures impinge on function, resulting in significant advances both scientif- ically and clinically.

• HSGAGs in Coagulation

Of the many functions regulated by HS- GAGs, one of the best studied is their Figure 1. HSGAGs at the cell surface modulate signaling pathways. HSGAGs exist at the cell role in the maintenance of hemostasis, surface as protein conjugates called proteoglycans. Due to the highly hydrophilic nature of the or regulation of blood volume and com- HSGAG chains, proteoglycans have a “Christmas tree”-like appearance. In addition, zooming position. In vivo, under normal, non- in on the HSGAG proteoglycans structure, the biosynthesis of HSGAGs leads to regions of ag- pathologic conditions, HSGAGs at the gregate chemical character in the polysaccharide chain. The darkened squares indicate under- surface of endothelial cells actively mod- sulfated regions of the polysaccharide chain, whereas the empty circles indicate regions of high sulfation. Zooming in further, all HSGAGs are comprised of a disaccharide unit of uronic ulate the activity of the proteases involved acid (either iduronic acid [I] or glucuronic acid [G]) attached to a hexosamine [H]. Each disac- in the coagulation cascade, including tis- charide unit can be differentially sulfated at the 2-O position of the uronic acid [2S] and/or the sue factor, factor Xa, and factor IIa 6-O [6S], 3-O [3S], and N [NS] positions of the hexosamine. The shorthand nomenclature is (thrombin), among others (Figure 2b) shown in brackets. Thus, the disaccharide structure in the ﬁgure is abbreviated GHNS,3S,6S rep- (Rosenberg 2001). As might be expected → resenting glucuronic acid 1 4 N-sulfoglucosamine 2,6-disulfate. from such an information dense molecule, HSGAGs impinge on almost every a hydration sphere to the vascular wall (Figure 2a). The disruption of this lining step of the coagulation cascade, mean- that is important for the normal tone by direct injury to the blood vessels is ing that these complex polysaccharides and ﬂuidity of the circulating media one of the primary initiating factors of play multifaceted roles in maintaining

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the interaction of HSGAGs with antithrombin III (ATIII) (Figure 3) (Jin et al. 1997). The entire coagulation process is under the control of a group of glyco- protein serine protease inhibitors (also known as serpins), the most important of which is ATIII. Many of the coagulation proteases, most notably factor Xa and IIa, are inhibited by ATIII, which forms an equimolar inhibitory complex with these enzymes. Much attention has focused on the binding of ATIII to HSGAGs; indeed, this interaction has become the prototypic example of a HSGAG–protein interaction. ATIII’s anticoagulant activity is mediated by binding to speciﬁc HSGAG sequences, viz., a pentasaccharide sequence within the

HSGAG chain with the sequence HNAc/S,6S GHNS,3S,6S I2SHNS,6S (Figure 3a) (Desai et al. 1998; Shriver et al. 2000). Binding of ATIII to this HSGAG pentasaccharide sequence at the cell surface of endothelial cells induces a conformational change in the protein, increasing by over three orders of magnitude its ability to inhibit selected proteases of the coagulation cascade, most notably factors Xa and IIa. Importantly, the interaction of ATIII with factor Xa occurs by a different mechanism than its interaction with factor IIa (Nugent 2000, Petitou et al. 1999). In each case, the first event tem- porally is the binding of ATIII to the pentasaccharide sequence followed by ATIII’s conformational change and con- Figure 2. HSGAGs at the endothelial cell surface actively participate in various aspects of cardiovascular biology. (A) HSGAGs mediate hemostasis by promoting the activity of such antico- comitant activation. For the inhibition agulant factors as ATIII. In addition, endothelial cell HSGAGs regulate lipid metabolism, pri- of factor Xa, this conformational change marily through binding apolipoproteins as well as lipoprotein lipase (LPL). Also, HSGAGs in ATIII is sufficient for the formation mediate the motility of blood cells, including platelets. Finally, the HSGAG coat of the endothe- of a high-affinity protein–protein inhib- lium provides a physical protective layer of negative charge, reducing the viscosity of the blood. itory complex (Jin et al. 1997). The (B) HSGAGs regulate the activity of a number of factors involved in the coagulation cascade. Xa:ATIII complex irreversibly traps the Shown are the multiple proteases involved in the coagulation process (all designated with a “F” protease as a reaction intermediate co- for factor, for example, FXI, FX, FVIII, etc.) that must be activated in turn (designated with an “a”). The coagulation cascade involves two arms, the intrinsic and extrinsic pathway, that con- valently bound to the serpin. Conversely, verge at the activation of factor X (to form FXa) as well as the penultimate step of the cascade, ATIII’s inhibition of thrombin requires a viz., the cleavage of prothrombin to form activated thrombin (also known as FIIa). Thrombin HLGAG chain containing at least 16 sac- then cleaves fibrinogen to form monomers that are able to aggregate and form a mesh that charide units (Figure 3b) (Petitou et al. leads to a clot. Several inhibitors of this cascade are known, including antithrombin III and 1999). Thrombin is thought to bind, tissue factor pathway inhibitor. These inhibit specific proteases of the coagulation cascade, des- through nonspecific ionic interactions, ignated in the figure with dashed arrows. Also shown are processes that inactivate proteases of the coagulation process, including activated protein C (shown by circles with dashes through to the nonreducing end of the chain. them). HSGAGs interact with a number of proteins to inhibit clot formation and promote clot Upon thrombin binding, activated ATIII clearance. These proteins, including ATIII, tissue factor pathway inhibitor and tissue plas- interacts with thrombin via the forma- minogen activator, are boxed. tion of a ATIII:IIa:HSGAG ternary complex. Thus, in this case, the HSGAG chain acts as a bridge or platform, promoting hemostasis, thus providing a potent ba- tion, preventing hypercoagulable states the formation of an ATIII:IIa high affin- sis for their pharmacological use (see and maintaining hemostasis (Figure 2b). ity complex (Figure 3b). below). Predominantly, HSGAGs serve One of the best recognized roles for In addition to the role of HSGAGs in as potent negative regulators of coagula- HSGAGs in the coagulation cascade is binding to ATIII and activating it, HS-

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are many and varied. Side effects such as heparin-induced thrombocytopenia are primarily associated with the long, poly- sulfated chain of pharmaceutical heparin, which provides binding domains for various proteins beyond what is therapeutically required (Baglin 2001). To circumvent the deleterious effects associated with heparin therapy while maintaining its potent anticoagulant activity, numerous strategies have been designed to create low molecular weight heparins (LMWHs), molecules with reduced chain length and, accordingly, reduced side effects. A number of strategies have been employed to create LMWHs, including controlled chemical cleavage (for instance, nitrous acid degradation or radical- mediated chain depolymerization), enzy- matic digestion, and size fractionation of heparin; each has met with a varying degree of clinical success (Casu and Torri 1999; Gunay and Linhardt 1999). LMWHs are currently defined as hav- Figure 3. HSGAGs regulate the activity of antithrombin III, a key inhibitor of the coagulation ing molecular masses of less than 6 kDa cascade. (A) Binding of a specific pentasaccharide sequence to ATIII with nanomolar affinity induces a conformational change in the protein, increasing by several orders of magnitude (compared to 9–12 kDa for UFH) and an ATIII’s anti-Xa and anti-IIa activity. Shown in black (and with arrows) are the modifications anti-Xa: anti-IIa ratio of 1.5:1 (again within the HSGAG polysaccharide chain that are most important for high affinity binding to compared to an anti-Xa to anti-IIa ratio ATIII, viz., an unsulfated glucuronic acid linked 14 to a trisulfated glucosamine that includes of approximately 1:1 for UFH) (Fareed rare 3-O sulfation. (B) Although the anti-Xa activity of HSGAGs is dependent only on ATIII et al. 2000). Of note is the fact that one (black diagram) binding to the pentasaccharide sequence (shown in black ovals) and undergo- of the consequences of the production ing a conformational change, its anti-IIa activity requires a longer chain (extension of white of various LMWHs is a reduction in the ovals) to which factor IIa (gray diagram) binds. In this case, the HSGAG acts a bridge promoting the formation of a ATIII:thrombin complex. amount of anti-IIa activity associated with the polysaccharide chain. This is because the length of LMWHs, as well GAGs are known to play other impor- used as highly effective antithrombotic as the placement within the polysaccha- tant roles in maintaining hemostasis. agents. Highly sulfated HSGAGs iso- ride chain of the ATIII binding site, pre- Recently, it has been found that HS- lated from mast cells have been used vents the efficient formation of an ATIII, GAGs actively facilitate the release of therapeutically for over 60 years as antithrombin, polysaccharide complex re- tissue factor pathway inhibitor (TFPI, coagulants. This highly sulfated popula- quired for the thrombin inhibition. As also known as extrinsic pathway inhibition of HSGAGs, termed “heparin,” is a such, current LMWHs are appropriate tor) (Fareed et al. 2000, Sandset et al. potent anticoagulant, used in a wide va- heparin substitutes in some clinical situ- 2000). TFPI is a polydomain inhibitor riety of thromboembolic disorders, in- ations, but not others. For instance, that inhibits tissue factor, factor VIIIa, cluding deep vein thrombosis, pulmo- LMWHs, with their pronounced ability factor Xa, and proteases released from nary embolism, arterial thromboses, and to inhibit factor Xa, have proven to be macrophages and leukocytes. Other im- acute coronary syndromes like myo- ideal agents for the treatment and pre- portant components of the coagulation cardial infarction and unstable angina vention of deep vein thrombosis but system that are modulated by HSGAGs (Antman 2001). Of note is the fact that not for arterial thrombosis, where anti- include heparin co-factor II (HCII) the multitude of clinical effects initiated thrombin activity is required. Recently, (Ramp et al. 2001, Nader et al. 2001) by heparin have not been recapitulated it has been noted that LMWHs differ and von Willebrand Factor (vWF) (Ant- with synthetic agents to date. from one another in terms of their anti- man and Handin 1998). In light of the Xa activity, bioavailability, and overall fact that a given HSGAG chain contains clinical efficacy (Jeske and Fareed 1999). • Creation of Low Molecular multiple distinct sequences, it seems However, owing to the complex chemi- Weight Heparins highly likely that these molecules play cal nature of LMWHs, differences in other roles in preventing blood coagu- Unfractionated heparin (UFH) has proven function cannot currently be attributed lation and maintaining the hemostatic to be a highly effective anticoagulant to differences in structure, a key link balance. and antithrombotic agent for a variety that would enable the production of a Given the multidimensional role of of clinical indications. However, the side new generation of LMWHs with im- HSGAGs, these molecules have been effects associated with heparin therapy proved clinical profiles.

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• Novel Technologies to Understand ease. Promising results have been ob- growth factor’s trajectory starting from LMWH Structure–Function tained in animal models where adminis- its release into the ECM until its binding tration of either FGF2 or VEGF increased at the cell surface of a recipient cell (Fig- Several novel technologies have been angiogenesis and the development of ure 4). Several mechanisms seem to be brought to bear on this important prob- new collateral vessels resulting in im- important in vivo. lem. Recently, a high throughput sequenc- proved perfusion and histology (Freed- ing strategy was developed for the struc- man and Isner 2001). However, there is • HSGAG Sequences in the ECM Seques- tural analysis of biologically and clinically a need to improve formulation, stability, ter FGF. HSGAG proteoglycans in the important HSGAGs (Turnbull, Powell, pharmacokinetics, and potency of these ECM, particularly in the basement and Guimond 2001; Venkataraman et al. angiogenic factors. A clear understand- membrane, can bind FGF and store it 1999). This technology combines state- ing of the modulatory role of cell surface in an inactive form (Chang et al. of-the-art analytical methodologies with HSGAGs in FGF and VEGF signaling will 2000). Degradation of these “storage” a bioinformatics framework to quickly provide a basis for designing more stable forms of HSGAGs results in the facile determine unknown sequences. With the and potent pro-angiogenic factors. release of active FGF, enabling a rapid use of this technology, it was found that Recent evidence has demonstrated response to external stimuli without certain means of degrading the heparin that specific HSGAG sequences modu- the need for new protein synthesis. chain to create LMWHs resulted in cleav- late the activity of FGF. Developmental • HSGAGs Form a Gradient Regulating age of the ATIII binding site (Shriver et al. studies in Drosophila have demon- FGF Diffusion from the ECM to the 2000). Furthermore, as predicted from strated that loss of specific HSGAG se- Cell Surface. Developmental studies in the structural analysis, it was found that quences results in morphogenic defects Drosophila have demonstrated that such strategies resulted in the genera- associated with inactivation of FGF- HSGAGs are one of the primary tion of LMWHs with lower activity in mediated signaling pathways (Lin et al. mechanisms enabling the formation vitro and in vivo compared to UFH 1999). Other studies have shown that of a concentration gradient for growth (Shriver, et al. 2000). Using such a strat- HSGAGs at the cell surface are neces- factors, cytokines, and other signaling egy, it should prove possible to generate sary for high-affinity binding of FGF, molecules (Perrimon and Bernfield LMWHs with increased activity and de- presentation of FGF to its transmem- 2000). Thus, by differential binding of creased side effects, thus creating a more brane receptor, and concomitant activa- FGF to HSGAGs in the ECM and at potent, more chemically defined second- tion of intracellular signaling pathways. the cell surface, the diffusion of FGF generation LMWH. becomes a highly regulated process thus controlling the rate and strength • Mechanism of HSGAG Action of FGF signaling. • HSGAGs as Modulators on FGF Activity • HSGAGs Mediate the Formation of an of Angiogenesis HSGAGs interact with growth factors Active Signaling Complex at the Cell In addition to the role of HSGAGs in like FGF at every known step of the Surface. FGF oligomerizes in the pres- modulating the coagulation process, these complex polysaccharides are known to be potent mediators of angiogenesis (Folkman and Shing 1992, Sasisekharan et al. 1994). As in hemostasis, HSGAGs play a multifaceted role in maintaining the angiogenic balance. However, unlike in the case of coagulation, where HS- GAGs largely prevent hypercoagulation, different HSGAG sequences can play distinct, even opposite, roles in the angiogenic process. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are two of the primary angiogenic factors that have been extensively charac- terized (Nugent and Iozzo 2000; Robin- Figure 4. HSGAGs regulate growth factor signaling in multiple ways in the molecule’s trajec- son and Stringer 2001). HSGAGs have tory from the originator cell to the recipient cell. (1) HSGAGs can sequester and store growth been shown to play essential roles in the factors (shown as white circles) in the ECM. (2) Upon degradation of the “storage” ECM, HS- angiogenic activities of both FGF and GAGs are involved in transport of growth factors to their target cells. (3) HSGAGs at the cell VEGF, particularly for FGF2 signaling, surface actively participate in the formation of an active signaling complex at the cell surface. where specific regulatory roles for HS- HSGAGs can facilitate the formation of an active signaling complex through either increasing the local concentration of the ligand at the cell surface, promoting ligand and/or receptor GAGs are known. Currently, both FGF2 dimerization, and/or stabilizing the ligand–receptor complex through bridging the ligand to and VEGF are being tested for their abil- the receptor. Formation of a signaling complex at the cell surface leads to activation of intra- ity to promote therapeutic angiogenesis cellular signaling pathways that in turn cause changes in cell phenotype, for example, cell pro- in cases of ischemic cardiovascular dis- liferation or cell migration.

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ence of specific HSGAG sequences and activity (Pye and Gallagher 1999). A cesses, including atherosclerosis and tu- (Kwan et al. 2001). Binding of FGF related approach is to dimerize peptides morigenesis (Sugahara and Kitagawa oligomers, mediated by cell surface with high affinity to an isoform of the 2000). Presently, transgenic animals are HSGAGs, to tyrosine kinase receptors FGF receptor, dramatically increasing becoming available with defined genetic results in increased receptor oligo- activity (Ballinger et al. 1999). An addi- changes in HSGAG biosynthesis, includ- merization, increased transphospho- tional approach that has met with a ing the absence of specific sulfotrans- rylation, and accordingly, increased higher degree of success is the creation, ferase genes (for instance, 2-O sulfotrans- activation of intracellular signaling via recombinant protein technology, of ferase or N-deactylase/N-sulfotransferase) pathways. In addition to mediating an oligomeric FGF construct, mimick- or specific protein core sequences of FGF oligomerization, HSGAGs can ing the role of HSGAGs in the forma- proteoglycans (for instance, glypican, syn- either promote or inhibit binding of tion of an FGF complex that binds with decan, or perlecan isoforms). Together, particular FGF family members to a high affinity to cell surface receptors. these transgenic animals should provide given receptor isoform (Berry et al. Recently, it has been shown that such a a solid foundation to further investigate 2001). As such, HSGAG sequences construct has a markedly increased the biological roles of specific HSGAG can act as molecular switches, modu- ability to induce angiogenesis in vivo structural motifs in many biological lating the affinity of a given FGF for (Kwan et al. 2001). The improved phar- processes. Second, recent advances in various receptor isoforms. macodynamics and pharmacokinetics the development of array technologies of engineered FGFs should prove useful will enable the rapid identification and Thus, HSGAGs, depending on sequence in the treatment of cardiovascular dis- sequencing of in vivo HSGAG structures and the biological context, can serve eases; for instance, a potent FGF may that bind with high affinity to given pro- multiple regulatory roles in FGF signal- expedite the process of angiogenesis to teins of therapeutic and/or biological ing. Importantly, FGF is used here as an improve the perfusion of ischemic importance (Keiser et al. 2001). Similar illustrative example (albeit an important myocardium. array technologies in proteomics have one); other growth factors and cytokines provided a key technology for the identi- that are currently used clinically bind to fication of proteins important in a bio- • Future Directions HSGAGs, including VEGF (as mentioned logical process. Finally, significant ad- above), interferon gamma, tissue plas- The structure–function paradigm outlined vances have been made in the synthesis minogen activator, and others. Each is in this review opens new approaches and of HSGAG sequences and HSGAG mi- regulated by HSGAGs of differing se- strategies for therapeutic intervention at metics, including the development of com- quence and via similar, but subtly differ- the cell–tissue–organ level. For example, binatorial libraries for the discovery and ent, mechanisms. Given the complexity an understanding of how HSGAGs, in a optimization of lead compounds (Petitou of the system the question then arises of dynamic fashion, impinge on the topolog- et al. 1999, Plante et al. 2001). Similar to how to translate this wealth of biologi- ical and functional organization of cells the solid-phase synthesis of DNA and cal information into therapeutically via- like smooth muscle cells and endothelial polypeptides that has enabled the bio- ble means of intervening in growth fac- cells can lead to important breakthroughs technology revolution, so should the de- tors’ signaling pathways. To this complex in heart and blood vessel regeneration. A velopment of rapid synthetic approaches problem, several novel technologies can sophisticated understanding of ECM–cell to oligosaccharide generation enable the be applied. First, as was the case with interactions will propel forward the field creation of new oligosaccharide-based the structural analysis of anticoagulant of organ repair and/or tissue engineering therapeutics. heparin, sequencing approaches prom- by permitting the targeting of a whole or- In short, a better understanding of the ise to shed some light on inhibitory and gan instead of a single cell. Also, identifi- role of HSGAGs is likely to become very activatory HSGAG sequences (Venka- cation of specific sequences that impinge important for the rapid development and taraman et al. 1999). Structural analy- on a particular biological process will al- expansion of the field of glycomics, or the sis of these sequences, in conjunction low for the development of novel molec- study of how proteins and saccharides with crystal structure analysis, should ular therapeutics based on polysac- interact with one another, thus imping- make possible the development of a charide structure. Synthetic molecular ing on cell physiology. Especially with completely novel set of growth factor mimics of HSGAG sequences may pro- the maturation of the field of genomics agonists and antagonists. In addition, vide new agents to combat diseases such and the sequencing of the human ge- given our understanding of certain as atherosclerosis, thromboembolic dis- nome, the next stage in obtaining a com- HSGAG–protein systems (for instance, the orders, and many others. plete understanding of how genotype FGF:HSGAG system), protein engineer- Three important advances, in addi- translates into phenotype is through the ing approaches have been applied to- tion to the newly devised sequencing ap- fields of proteomics and glycomics. wards the development of clinical prep- proaches outlined above, make such a arations of growth factors like FGF with vision realizable. First, the recent clon- • Acknowledgments higher activity and less variable phar- ing of many of the genes involved in the macokinetics. Three distinct methodolo- biosynthesis of HSGAGs and HSGAG We would like to thank Dr. Ganesh gies have been employed. The first is to proteoglycans promises to provide a ba- Venkataraman for help with this manu- create a covalent FGF–HSGAG com- sis for the dramatic increase in our ap- script. Financial support from the follow- plex using a defined HSGAG sequence preciation of the role of HSGAGs in ing funding agencies is acknowledged: the that is known to promote FGF signaling modulating fundamental biological pro- National Institutes of Health (GM 57073,

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