Glycosaminoglycan Synthases: Catalysts for Customizing Sugar

Downloaded by MICHIGAN STATE UNIV on August 21, 2013 | http://pubs.acs.org Publication Date (Web): August 11, 2010 | doi: 10.1021/bk-2010-1043.ch021

Chapter 21

Glycosaminoglycan Synthases: Catalysts for Customizing Sugar Polymer Size and Chemistry Paul L. DeAngelis* Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma Center for Medical Glycobiology, 940 S.L. Young Blvd., Oklahoma City, OK 73126, USA *[email protected]

Synthesis of sugar polymers has always been a challenge. Total organic chemistry approaches are appropriate for smaller oligosaccharides (less than 6 monosaccharides), but as the chain length increases, the efficiency and yields decrease while the production of non-target compounds increases. In addition, typical carbohydrate chemistry results in much waste solvent and spent toxic reagents. To assist the chemist, enzymes, in particular glycosyltransferases and hydrolases, have been employed with success to make natural and artificial structures. Enzyme catalysts have high efficiency, great stereo-selectivity and regio-specificity, and usually operate in aqueous (‘green’) systems. Here, the production of a variety of short (5 monosaccharides) to long (~12,000 monosaccharides) monodisperse heteropolymers with many potential medical applications using biosynthetic enzymes is described.

Introduction Pasteurella multocida bacteria produce extracellular capsules composed of the glycosaminoglycans [GAGs] hyaluronan, chondroitin, and heparosan (1). These linear polysaccharides with repeating [GlcA-HexNAc] disaccharide subunits also form the backbones of polymers in many vertebrate tissues. The bacterial GAG capsules are virulence factors that allow the microbes to be more successful pathogens by acting as molecular camouflage that render host © 2010 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


defenses less effective. We have harnessed several bacterial GAG synthases, the bifunctional enzymes that polymerize the GAG chains using UDP-sugar precursors (Figure 1), for chemoenzymatic synthesis in vitro to make novel GAGs with potential for a variety of medical applications. Our biotechnology focus is on drug delivery (both bio-inert stealthy or targeted vehicles) and biomaterial platforms (implantable gels and cell scaffolds). Key knowledge for developing these new GAG polymer production systems was the identification of the GAG synthases, the dual-action enzymes that polymerize the GAG chains using both UDP-GlcUA and UDP-HexNAc precursors according to the following reaction:

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

Experimental Chemoenzymatic Synthesis of Polymers Reactions containing UDP-sugars (natural or synthetic), an acceptor (either native or biotin-derivatized GAG oligosaccharides or synthetic glycosides), and GAG synthase enzymes (either recombinant maltose-binding protein-PmHS fusions (2) or PmHAS (1–3) truncations (3) were combined in liquid phase reactions in a similar fashion to our previous reports. The products were analyzed by agarose gel electrophoresis with Stains-all detection, polyacrylamide gels with Alcian Blue detection, mass spectroscopy and/or gel filtration chromatography coupled to a light scattering detector.

Results and Discussion Naturally 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 five monosaccharides, are also difficult to synthesize by strictly organic synthesis. In contrast, chemoenzymatic synthesis in vitro offers the potential to harness enzyme catalysts for rapid, efficient reactions. 300 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. Some useful GAG synthases are the Pasteurella enzymes that make HA, PmHAS, and heparosan, PmHS1 or PmHS2. Even though the same monosaccharides are transferred during HA and heparosan biosynthesis, the glycosidic linkages are 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). Mutagenesis of one active site often leaves the remaining active site unperturbed thus creating new catalysts for step-wise reactions rather than polymerization. The synthase 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, DNA standards; 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 from Streptococcus 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) GAG polysaccharides of a desired size by controlling stoichiometry in synchronized reactions (Figure 2) (3). The use of an acceptor (a short GAG chain that mimics 301 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 the random, slow initiation of the de novo synthesis thus all GAG chains are elongated in 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 is not employed, a wide variety of chain sizes (i.e. polydisperse) are formed from asynchronous elongation events. We have also created GAG oligosaccharide synthesis systems employing immobilized mutant enzyme reactors in a step-wise sugar addition strategy; pentamers to 22-mers have been prepared (4). A normally bifunctional enzyme is mutated to inactivate one of the transferase activities, but the other transferase remains functional. In an example of the oligosaccharide synthesis strategy, a mutant catalyst (e.g., a GlcNAc-tase) is used to transfer a single sugar to an acceptor (e.g, a chain terminating with a GlcUA unit), and then the reaction mixture is removed and allowed to react with the next mutant catalyst (e.g., a GlcUA-tase). The strategy may be repeated to build a variety of GAG polymers. Certain GAG synthases exhibit relaxed acceptor specificity allowing non-cognate molecules to be elongated. For example, the creation of hybrid or chimeric sugar molecules containing both HA-like and chondroitin-like disaccharide repeats have been prepared. In our most recent work, we have expanded the GAG chemical functionality repertoire. Our main approach is to make synthetic UDP-sugars containing unnatural substitutions (including azido, alkyne, alkyl, fluoro, or protected amine groups) 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 strategy and the reaction conditions, we have made a variety of GAG-like polymers from 5 to ~10,000 sugars where either one or multiple artificial sugar units are added to a single chain. The new groups in the GAG chain promise to enhance their potential 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 using UGP-GlcN[TFA] with PmHAS enzyme (middle) and the de-protected pentamer with a free amine (bottom). 302 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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


Acknowledgments The author of this paper would like to thank the various researchers who contributed 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, the Oklahoma Center for the Advancement of Science and Technology, and NSERC of Canada, provided financial support of this research.

References 1. 2. 3. 4.

DeAngelis, P. L. Glycobiology 2002, 12, 9R. Sismey-Ragatz, A. E.; Green, D. E.; Otto, N. J.; Rejzek, M.; Field, R. A.; DeAngelis, P. L. J. Biol. Chem. 2007, 282, 28321. Jing, W.; DeAngelis, P. L. J. Biol. Chem. 2004, 279, 42345. DeAngelis, P. L.; Oatman, L. C.; Gay, D. F. J. Biol. Chem. 2003, 278, 35199.

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

Glycosaminoglycan Synthases: Catalysts for Customizing Sugar

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