Chapter 21

Glycosaminoglycan Synthases: Catalystsfor Customizing Sugar Polymer Size and

Chemistry

Paul L. DeAngelis*

Dept. of Biochemistry and Molecular Biology, University of OklahomaHealth Sciences Center, Oklahoma Center for Medical Glycobiology,

940 S.L. Young Blvd., Oklahoma City, OK 73126, USA*paul-deangelis@ouhsc.edu

Synthesis of sugar polymers has always been a challenge.Total organic chemistry approaches are appropriate for smalleroligosaccharides (less than 6 monosaccharides), but as thechain length increases, the efficiency and yields decreasewhile the production of non-target compounds increases.In addition, typical carbohydrate chemistry results in muchwaste solvent and spent toxic reagents. To assist the chemist,enzymes, in particular glycosyltransferases and hydrolases,have been employed with success to make natural and artificialstructures. Enzyme catalysts have high efficiency, greatstereo-selectivity and regio-specificity, and usually operate inaqueous (‘green’) systems. Here, the production of a variety ofshort (5 monosaccharides) to long (~12,000 monosaccharides)monodisperse heteropolymers with many potential medicalapplications using biosynthetic enzymes is described.

Introduction

Pasteurella multocida bacteria produce extracellular capsules composedof the glycosaminoglycans [GAGs] hyaluronan, chondroitin, and heparosan(1). These linear polysaccharides with repeating [GlcA-HexNAc] disaccharidesubunits also form the backbones of polymers in many vertebrate tissues. Thebacterial GAG capsules are virulence factors that allow the microbes to bemore successful pathogens by acting as molecular camouflage that render host

© 2010 American Chemical Society

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defenses less effective. We have harnessed several bacterial GAG synthases,the bifunctional enzymes that polymerize the GAG chains using UDP-sugarprecursors (Figure 1), for chemoenzymatic synthesis in vitro to make novel GAGswith potential for a variety of medical applications. Our biotechnology focus ison drug delivery (both bio-inert stealthy or targeted vehicles) and biomaterialplatforms (implantable gels and cell scaffolds).

Key knowledge for developing these new GAG polymer productionsystems was the identification of the GAG synthases, the dual-action enzymesthat polymerize the GAG chains using both UDP-GlcUA and UDP-HexNAcprecursors according to the following reaction:

Sugar polymers, especially molecules with chain lengths longer than fivemonosaccharides, are difficult to produce by strictly organic synthesis in amonodisperse, defined form as well as usually generate ~1,000:1 waste to targetmolecules. ‘Green’ chemoenzymatic synthesis offers the potential to harnessenzyme catalysts for rapid, efficient reactions. We have developed methods toconstruct GAG polysaccharides of any desired size from 10 to 25,000 kDa insynchronized reactions as well as make short GAG oligosaccharides (0.8 to 5 kDa)in step-wise addition reactions. We have also enhanced the potential syntheticrepertoire by employing novel UDP-sugars that allow further functionalization ofGAGs. For example, a variety of new polymers with unnatural chemical groupsin various positions (either single or multiple novel sugar units) have now beenmade facilitating coupling reactions including ‘click’ chemistry.

ExperimentalChemoenzymatic Synthesis of Polymers

Reactions containing UDP-sugars (natural or synthetic), an acceptor (eithernative or biotin-derivatized GAG oligosaccharides or synthetic glycosides), andGAG synthase enzymes (either recombinant maltose-binding protein-PmHSfusions (2) or PmHAS (1–3) truncations (3) were combined in liquid phasereactions in a similar fashion to our previous reports. The products were analyzedby agarose gel electrophoresis with Stains-all detection, polyacrylamide gels withAlcian Blue detection, mass spectroscopy and/or gel filtration chromatographycoupled to a light scattering detector.

Results and DiscussionNaturally occurring polysaccharides and oligosaccharides from various

organisms are often difficult to prepare in a pure, defined, monodisperse form.Sugar polymers, especially molecules with chain lengths longer than fivemonosaccharides, are also difficult to synthesize by strictly organic synthesis.In contrast, chemoenzymatic synthesis in vitro offers the potential to harnessenzyme catalysts for rapid, efficient reactions.

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Figure 1. Schematic of Recombinant PmHAS and PmHS Enzymes. Someuseful GAG synthases are the Pasteurella enzymes that make HA, PmHAS, andheparosan, PmHS1 or PmHS2. Even though the same monosaccharides aretransferred during HA and heparosan biosynthesis, the glycosidic linkagesare different and the PmHAS or PmHS protein sequences are not very similar.Each polypeptide chain contains two relatively independent glycosyltransferase(Tase) activities (each with an acceptor site and a donor site). Mutagenesisof one active site often leaves the remaining active site unperturbed thus

creating new catalysts for step-wise reactions rather than polymerization. Thesynthase proteins will catalyze GAG synthesis in vivo or in vitro as long as the

UDP-sugars are supplied.

Figure 2. Agarose gel analysis of monodisperse HA Polymers. D, DNAstandards; Sm, mixture of five synthetic HAs ranging from 1,500 to 495 kDa ;2.4 MDa synthetic HA, N, natural HA from rooster comb; N’, natural HA fromStreptococcus bacteria; Sm’, mixture of five synthetic HAs ranging from 495 to

27 kDa.

We have developed methods to construct large (0.01 to 8,000 kDa) GAGpolysaccharides of a desired size by controlling stoichiometry in synchronizedreactions (Figure 2) (3). The use of an acceptor (a short GAG chain that mimics

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

the nascent polymer terminus) to prime the GAG synthase circumvents therandom, slow initiation of the de novo synthesis thus all GAG chains are elongatedin parallel and achieve the same size (i.e., nearly monodisperse; polydispersity= 1.02-1.2 depending on final polymer size). In contrast, when the acceptor isnot employed, a wide variety of chain sizes (i.e. polydisperse) are formed fromasynchronous elongation events.

We have also created GAG oligosaccharide synthesis systems employingimmobilized mutant enzyme reactors in a step-wise sugar addition strategy;pentamers to 22-mers have been prepared (4). A normally bifunctional enzymeis mutated to inactivate one of the transferase activities, but the other transferaseremains functional. In an example of the oligosaccharide synthesis strategy, amutant catalyst (e.g., a GlcNAc-tase) is used to transfer a single sugar to anacceptor (e.g, a chain terminating with a GlcUA unit), and then the reactionmixture is removed and allowed to react with the next mutant catalyst (e.g., aGlcUA-tase). The strategy may be repeated to build a variety of GAG polymers.Certain GAG synthases exhibit relaxed acceptor specificity allowing non-cognatemolecules to be elongated. For example, the creation of hybrid or chimeric sugarmolecules containing both HA-like and chondroitin-like disaccharide repeatshave been prepared.

In our most recent work, we have expanded the GAG chemical functionalityrepertoire. Our main approach is to make synthetic UDP-sugars containingunnatural substitutions (including azido, alkyne, alkyl, fluoro, or protected aminegroups) that the GAG synthases can recognize and incorporate into sugar polymers(Figure 3). Most analogs do not work as well as the authentic natural precursors,but a few are even better substrates. By manipulating the sugar addition strategyand the reaction conditions, we have made a variety of GAG-like polymers from5 to ~10,000 sugars where either one or multiple artificial sugar units are addedto a single chain. The new groups in the GAG chain promise to enhance theirpotential for use in chemical reactions and/or possess new biological activities.

Figure 3. Mass spectrometric analyses of a HA tetramer tagged with a fluorescein(top), the protected amine (GlcN[TFA]) addition pentamer product made usingUGP-GlcN[TFA] with PmHAS enzyme (middle) and the de-protected pentamer

with a free amine (bottom).

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Conclusion

The study of the GAG synthases is passing its infancy, but as more knowledgeis gained, the production of new generation GAG-like polymers with betterbiological and/or chemical properties is expected. Some of the expected novelGAG therapeutics include safer anti-coagulants, biomaterials that gel afterinjected into the body, and improved non-toxic drug delivery systems.

Acknowledgments

The author of this paper would like to thank the various researchers whocontributed to this work including: Dixy E. Green, Nigel J. Otto, F.Michael Haller,Wei Jing, Alison E. Sismey, Regina C. Visser, Robert J. Linhardt, Michel Weiwer,Martin E. Tanner and Gert-Jan Boons. The National Institutes of Health, theOklahoma Center for the Advancement of Science and Technology, and NSERCof Canada, provided financial support of this research.

References

1. DeAngelis, P. L. Glycobiology 2002, 12, 9R.2. Sismey-Ragatz, A. E.; Green, D. E.; Otto, N. J.; Rejzek, M.; Field, R. A.;

DeAngelis, P. L. J. Biol. Chem. 2007, 282, 28321.3. Jing, W.; DeAngelis, P. L. J. Biol. Chem. 2004, 279, 42345.4. DeAngelis, P. L.; Oatman, L. C.; Gay, D. F. J. Biol. Chem. 2003, 278, 35199.

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In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.