Sugar Polymer Engineering with Glycosaminoglycan Synthase Enzymes

Chapter 16

Downloaded by OHIO STATE UNIV LIBRARIES on September 7, 2012 | http://pubs.acs.org Publication Date: February 15, 2005 | doi: 10.1021/bk-2005-0900.ch016

Sugar Polymer Engineering with Glycosaminoglycan Synthase Enzymes: 5 to 5,000 Sugars and a Dozen Flavors Paul L. DeAngelis Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Sciences Center, Oklahoma City, OK 73104

Glycosaminoglycans [GAGs] are used currently as therapeutics, but the controlled manipulation and/or synthesis of these sugars has been difficult or impossible in the past. A variety of GAG polysaccharides and oligosaccharides may now be synthesized in vitro with the advent of recombinant Pasteurella multocida synthases. Narrow size distribution polymers ranging from 5 to 1,500 kDa are produced with synchronized, stoichiometrically-controlled reactions. Alternatively, monodisperse oligosaccharides ranging in size from 5 to 22 monosaccharides are synthesized in a step-wise fashion with immobilized enzyme reactors. Either pure or chimeric sugar chains are possible in both types of synthetic schemes. Overall, these strategies should allow the production of a wide variety of novel polymers with potential utility in the treatment of cancer, wound-healing, thrombosis, immune dysfunction, or inflammation.

232

© 2005 American Chemical Society In Polymer Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

233


Introduction Glycosaminoglycans [GAGs] are linear heteropolysaccharides composed of repeating disaccharide units containing a derivative of an amino sugar (either glucosamine or galactosamine). Hyaluronan [HA], chondroitin, and Nacetylheparosan (or heparosan) contain glucuronic acid [GlcUA] as the other component of the disaccharide repeat. These sugar polymers are essential for vertebrate life playing many roles as structural elements and recognition or adhesion signals. Pathogenic bacteria often use a polysaccharide capsule, an extracellular sugar polymer coating surrounding the microbial cell, to surmount host defenses (1). A few bacteria synthesize GAG capsules that contribute to their virulence because the host and microbial polymers are chemically identical or similar and thus are relatively non-immunogenic (2). In addition to this molecular camouflage strategy, another emerging scenario is that these bacteria may also be able to commandeer vertebrate systems that employ GAGs. We have utilized the enzymes derived from a certain bacterial species, Pasteurella multocida, with favorable properties to create biomaterials suitable for a variety of health applications. Our chemoenzymatic methodologies allow the preparation of either defined oligosaccharides or narrow size distribution polysaccharides with a variety of GAG compositions. Three of the most prominent capsular types of P. multocida, a widespread Gram-negative animal bacterial pathogen, are sources of acidic GAGs. Carter Types A, D, or F produce hyaluronan, heparosan, or chondroitin polysaccharides, respectively (Table 1).

Table 1. Pasteurella GAGs and Synthases. Polysaccharide

Hyaluronan, HA Chondroitin (unsulfated) Heparosan (unsulfated)

Repeat Structure

Capsule enzyme Type

$4GlcUA- p3GlcNAc] $4GlcUA- P3GalNAc] [p4GlcUA-a4GlcNAc]

A F D

pmHAS pmCS pmHS

In Polymer Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

ref.

(5) (4) (5)

234 The various Pasteurella glycosyltransferases, called G A G synthases (25), required for the production of the G A G chain transfer two distinct monosaccharides to the growing chain in a repetitive fashion according to the overall reaction: η UDP-GlcUA + η UDP-HexNAc -»


In UDP + [GlcUA-HexNAcL

(1)

where HexNAc = N-acetylglucosamine [GlcNAc] or N-acetylgalactosamine [GalNAc]. Depending on the specific G A G and the particular organism examined, the degree of polymerization, n, ranges from ~10 " . The bacterial polymers are not further modified in contrast to the vertebrate systems where chondroitin and heparosan are found most often in a sulfated and/or epimerized state. 2

4

Escherichia co/i-derived recombinant pmHAS, pmCS, and pmHS will elongate exogenously supplied GAG-polymer acceptors in vitro (4-6). The HexNAc-transferase or the GlcUA-transferase activities of the Pasteurella enzymes can be assayed separately in vitro by supplying the appropriate acceptor oligosaccharide and only one of the UDP-sugar precursors. Single monosaccharides are added to the growing chain sequentially to the nonreducing reducing terminus of the linear polymer chain (6). The intrinsic fidelity of each transfer step assures the production of the GAG repeat structure. The reducing end of the acceptor may be derivatized with another molecule (e.g. a tag or a drug) or immobilized on a surface (e.g. glass, metal, or plastic) (7). Two tandemly repeated sequence elements are present in the pmHAS and pmCS polypeptides (Figure 1). Each element contains a set of two short sequence motifs: a DGS followed by a DXD (X = S or C) about 45 residues downstream. Mutation of the aspartate residue in any one DGS or DXD motif of pmHAS converts the dual-action synthase into a single-action glycosyltransferase (8,9). The GlcNAc-transferase and the GlcUA-transferase activities are relatively independent based on kinetic comparisons of the various mutants to the wild-type pmHAS enzyme. The Pasteurella chondroitin synthase, pmCS, contains separate GalNAc-transferase (a slightly mutated version of the GlcNAc-site of pmHAS) and GlcUA-transferase sites (4). A further practical improvement of pmHAS or pmCS is the conversion of the native sequence membrane proteins into soluble enzymes by the removal of the carboxylterminal membrane association domain (4,8). The Pasteurella heparosan synthase, pmHS, also appears to contain at least two domains, but it is not similar at the amino acid level to pmHAS or pmCS (5).


235

pmHAS

W/////M

DGS DCD

GlcNAc-Tase

GlcUA-Tase


pmCS

DGS DCD

GalNAc-Tase

GlcUA-Tase

Figure 1. Schematic Model of Domain Structure ofpmHAS and pmCS. These enzymes contain two distinct catalytic sites (important amino acid residues depicted in single letter code) responsible for transfer of the HexNAc or the GlcUA monosaccharide. Certain regions are dispensable for glycosyltransferase activity (hatched area). We have created recombinant versions of the Pasteurella pmHAS and pmCS enzymes that possess attractive features for chemoenzymatic synthesis including: fast reaction rates in vitro, good solubility and stability properties, and the lack of strong feedback inhibition by UDP (the byproduct of polymerization) in addition to their essential property of elongating exogenously supplied oligosaccharides. Furthermore, the mutant single-action enzymes that possess only one of the two component transferase activities (i.e. either a HexNActransferase or a GlcUA-transferase) allow stepwise synthesis strategies as in Equation 2 or 3; the mutant enzymes cannot repetitively polymerize both monosaccharides of the disaccharide repeat. UDP-HexNAc + [GlcUA-HexNAcL -> UDP + HexNAc-iGlcUA-HexNAcL

(2)

UDP-GlcUA + [HexNAc-GlcUAL -> UDP + GlcUA-[HexNAc-GlcUAL


(3)

236

Experimental Procedures

Enzymes and Reactors


1

703

1

704

The soluble, truncated dual-action pmHAS " or dual-action pmCS " enzyme were used for long chain polysaccharide synthesis in synchronized reactions. A pair of single-action enzymes: the GlcNAc-Tase pmHAS " (D527N,D529N) and the GlcUA-Tase pmHAS (D247N,D249N) were used in an immobilized state for stepwise oligosaccharide synthesis. The analogous chondroitin synthase-based GalNAc-Tase mutant, pmCS (D520N,D522N), was also immobilized. Mutants with two lesions per motif are preferable to mutants with single lesions when reactions employ high levels of UDP-sugars (70). The enzyme reactors (-18 mg protein on 4 ml of packed beads in a small glass column) were catalyttcally active for months with storage at 4°C in 50 mM Tris, pH 7.2,1 M ethylene glycol buffer (TEG) buffer. 1

703

1703

1 704

Synchronized, Stoichiometrically-controlled Polysaccharide Synthesis The synchronized syntheses in general contained a soluble dual-action synthase (pmHAS or pmCS as desired), a GAG-based acceptor, UDP-HexNAc (UDP-GlcNAc or UDP-GalNAc as needed), UDP-GlcUA, and 5 mM MnCl in TEG reaction buffer. Reactions are incubated at 30°C for 6 to 48 hrs. The HA tetrasaccharide, the starting acceptor for the synthesis of longer HA chains, was generated by exhaustive degradation of H A polymer with testicular hyaluronidase. Alternative acceptors included various chondroitin polymers (unsulfated or sulfate A, B , or C). The sizes of GAG polysaccharides were analyzed on agarose gels in l x TAE buffer and detected with Stains-All dye as described previously (77). Size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS) analysis was used to determine the absolute molecular weights of the polymers. Polymers were separated on two tandem Toso Biosep TSK-GEL columns (6000PWXL followed by 4000PWXL) eluted in 50 mM sodium phosphate, 150 mM NaCl, pH 7 at 0.5 mL/min. The eluant flowed through an Optilab DSP interferometric refractometer and then a Dawn DSF laser photometer (632.8 nm; Wyatt Technology, Santa Barbara, CA) in the multi-angle mode. The manufacturer's software package was used to determine the absolute average molecular weight using a dn/dC coefficient of 0.153. 2


237

Stepwise Oligosaccharide Synthesis with Enzyme Reactors In the typical oligosaccharide synthesis, 90 μιηο1β8 of acceptor oligosaccharide and 110-135 μ ι η ο ^ (1.2 to 1.5 equivalents) of UDP-sugar (-15 mM final) in reaction buffer (TEG plus 17 mM MnCl ) were circulated over an enzyme reactor at room temperature. For converting the HA tetrasaccharide starting material (with a GlcUA at the nonreducing terminus) into the pentasaccharide, the GlcNAc from UDP-GlcNAc was transferred with the GlcNAc-Tase reactor (~1 to 2 hours) as in Equation 2. The next UDP-sugar (in this specific case, UDP-GlcUA) was added to the reaction mixture and then applied to the next reactor (converting pentasaccharide into the hexasaccharide with immobilized GlcUA-Tase) to perform the reaction of Equation 3. This repetitive synthesis was continued by adding the next appropriate UDP-sugar and switching enzyme reactors. The reactions were monitored by thin layer chromatography (silica TLC plates developed with rc-butanol/acetic acid/H 0, 1.5:1:1 or 1:1:1) and napthoresorcinol staining (0.2% w/v in 96% ethanol/4% sulfuric acid; 100°C). For mass spectrometry (MALDI-TOF MS), the matrix solution (50 mg/ml 6-aza2-thiothymine in 50% acetonitrile, 49.9% water, 0.1% trifluoroacetic acid, 10 mM ammonium citrate) was mixed 1:1 with the samples containing -0.1 μg/μl oligosaccharide in water, spotted onto the target plate, and vacuum dried. The samples were analyzed in the negative ion, reflectron mode on a Voyager Elite DE mass spectrometer.


2

2

Results and Discussion The recombinant Pasteurella synthases produced in the appropriate bacterial host will elongate certain exogenously supplied glycosaminoglycan acceptor chains (4-6). Specifically, if the host bacterium does not produce one of the required sugar precursors, UDP-GlcUA (i.e. lacks the gene for UDPglucose dehydrogenase), then the recombinant synthase will not have an endogenous nascent GAG chain upon isolation from the cell. These virgin catalysts probably have open or available active sites that readily bind and then extend the exogenously supplied acceptor chain. For example, recombinant pmHAS will readily add sugars onto HA tetrasaccharides to create longer polymer chains in the range of 5 to 2,000 kDa. The recombinant pmHAS and pmCS enzymes appear to function in a non-processive fashion in vitro; the GAG chain is bound, extended, and released repeatedly during the polymerization process.


238


Model for Monodisperse HA Production The recombinant pmHAS synthesizes H A chains in vitro if supplied with both required UDP-sugars in a suitable reaction buffer. If recombinant pmHAS is supplied a HA-like oligosaccharide in vitro, then the overall incorporation rate is elevated up to 20- to 60-fold (6). It was suggested that the rate of initiation of a new HA chain de novo was slower than the subsequent elongation (Le. repetitive addition of sugars to a nascent HA molecule). Therefore, the observed stimulation of synthesis by exogenous acceptor appears to operate by bypassing the kinetically slower initiation step allowing the reaction in Equation 4 to predominate. We now further hypothesize that the polymerization by pmHAS in the presence of HA acceptor is a synchronized process in vitro and thus a more uniform defined HA should be obtained; all chains will be elongated in parallel resulting in a more homogenous final population. fast ->

η UDP-GIcUA + η UDP-HexNAc + [GlcUA-HexNAc]*

In UDP + [GlcUA-HexNAcU,

(4)

Model for Controlling Size of HA Products As noted above, chain initiation appears to be the rate-limiting step for pmHAS-catalyzed HA production. Due to this kinetic phenomenon, the synthase will add all available UDP-sugar precursors to the acceptor termini before much new chain initiation occurs. If the polymerization is indeed a synchronized process, then the amount of acceptor should affect the final size of the HA product when a limited amount of UDP-sugar is present. If there are many termini (Le. Eq. 5 where ζ is large), then the available UDP-sugars will be distributed among many molecules and thus result in many short polymer chain extensions. Conversely, if there are few termini (Le. Eq. 5 where ζ is small), then the available UDP-sugars will be distributed among a few molecules and thus result in a few long chain extensions.

η UDP-GIcUA + η UDP-HexNAc + ζ [GIcUA-HexNAc]

x

fast -»

In UDP + ζ [GlcUA-HexNAcL /z> +rn


(5)


239

To test our speculation, we performed a series of assays utilizing various levels of HA tetrasaccharide with a fixed amount of UDP-sugar and pmHAS. The size and polydispersity of the HA products was analyzed by SEC-MALLS (Table 2). In this example, decreasing amounts of tetrasaccharide (sample #1 > #5) were employed. As predicted, higher acceptor/UDP-sugar ratios resulted in shorter products. With this general strategy, we were able to generate HA from 27 kDa to 1.3 MDa with polydispersity ranging from 1.001 to 1.2 (72). For reference, a polydispersity value of 1 corresponds to an ideal monodisperse polymer. By calculating the ratio of acceptor to UDP-sugar, it is possible to select the final HA size desired. Overall, the products of synchronized, stoichiometrically-controlled reactions exhibit much more narrow size distributions in comparison to commercially available HA preparations from natural sources including chicken or bacteria (Fig. 2). The utility of HA preparations with a narrow size distribution should be extensive because many biological phenomena (e.g. angiogenesis, cell proliferation and signaling) respond differentially depending on the molecular weight of HA (13). Our preliminary studies of in vitro syntheses with pmCS suggest that a wide variety of monodisperse chondroitin polymers may also be synthesized.

Table 2. SEC-MALLS analysis of HA produced via synchronized, stoichiometrically-controlled reactions.

Sample

M

n

283,000 346,000 422,000 490,000 570,000

#1 #2 #3 #4 #5

M

w

284,000 347,000 424,000 493,000 575,000

polydispersity (My/M„) 1.001 1.002 1.004 1.006 1.010

M = number average molecular weight M = weight average molecular weight n

w



240

Figure 2. Agarose gel analysis of various monodisperse products of synchronized, stoichiometrically-controlled reactions. S = a mixture of the HA polymer products (1.3, 0.9, 0.6, 0.3, 0.027 MDa from top to bottom) from 5 different synchronized reactions loaded in 1 lane; Commercial HA = 4 different HA preparations; D = DNA standard ('kilobase ladder' -12 kb at top).


241

Production of Chimeric GAG Chains


We recently found that the various Pasteurella GAG synthases will also use non-cognate acceptor molecules (14). For example, we added a HA chain onto an existing chondroitin sulfate chain using pmHAS in Figure 3. This particular family of hybrid molecules may be useful as artificial proteoglycans for tissue engineering that do not contain the protein component of the natural cartilage proteoglycans.

Figure 3. Agarose gel analysis of HA-chondroitin hybrid polymers. HA chains of increasing length were added onto chondroitin sulfate A (CS-A) using pmHAS resulting in new bands with lower mobility. (D = DNA standard ladder. Arrow marks the position of the CS-A starting material)


242


Chemoenzymatic Synthesis of Defined Oligosaccharides The Pasteurella HA synthase, a dual-action polymerizing enzyme that normally elongates HA chains rapidly, possesses two active sites (5,9). Single sugars are added to the growing chain sequentially to the non-reducing terminus allowing the step-wise synthesis of oligosaccharides, if desired. We used mutagenesis to convert the native enzyme into two single-action glycosyltransferases. The resulting GlcUA-transferase and GlcNAc-transferase are appropriate for producing short sugar chains in a controlled, stepwise fashion without purification of the intermediates. For example, in Figure 4, in the synthesis converting the H A tetrasaccharide into a H A tetradecasaccharide, HAll-mer was converted into HA12-mer in one, quantitative step with an immobilized enzyme reactor.

HA12

70000

I

40000 2114.69

1000

1500

2000 Mass (m/z)

HAH

2500

Figure 4. Matrix-assisted laser desorption ionization time-of-flight spectrum of crude reaction mixtures from the oligosaccharide synthesis reactions. The mass of the H+ form of the target sugar is indicated; the train of higher mass ions are forms with increasing amounts ofNa+ (22 additional amu in comparison to H+) on the carboxylates ofGlcUA residues.


243


We have produced oligosaccharides each with a single length in the size range of 10 to 22 monosaccharides in a few days (70,75). Only simple desalting at the end of the reaction is required to purify the target sugar. The experimental masses and the predicted masses of these synthetic oligosaccharides were in excellent agreement. These small HA oligosaccharides have biological activities (e.g. inducing cancer cells to undergo apoptosis or stimulating angiogenesis) distinct from long polysaccharides (75). Due to the relaxed acceptor polymer specificity of pmHAS and pmCS, oligosaccharides with a mixed G A G structure may be synthesized by employing different HexNAc-transferase reactors during the synthesis (75). For example, Figure 5 depicts the TLC analysis of the HA-chondroitin octasaccharide, [GlcUA-GalNAc] [GlcUA-GlcNAc] ; this hybrid compound is isobaric with the HA octasaccharide (Le. the epimers GlcNAc and GalNAc have same mass), but normal phase chromatography can distinguish the two sugars. 2

2

Figure 5. TLC analysis of HA-chondroitin octasaccharide. The novel sugar (marked with star) migrates slower than the pure HA octasaccharide (HA 8).


244


Current Research Aims We have also created new chimeric enzymes (e.g. a polypeptide composed of segments of both pmHAS and pmCS) that have reduced selectivity for the sugar transfer reaction (77). Normally, native sequence pmHAS will only transfer UDP-GlcNAc while native sequence pmCS will only transfer UDPGalNAc; relatively strict donor specificity is observed. On the other hand, certain artificial synthase constructs will transfer either natural UDP-hexosamine or the unnatural substrate UDP-GlcN. Thus, depending on the UDP-sugar precursors supplied, one can produce novel GAGs containing (i) a blended mixture of HA and chondroitin or (li) GAGs with unnatural functionalities such as free amino groups. In the former case, the new polymer will have some attributes of both GAGS. In the latter case, the new reactive functionalities on the polymer chain are useful for creating cross-linked viscoelastic gels or polyvalent drug-carriers. The full scope and range of the artificial synthases' catalytic prowess is a subject of current investigation. Other new catalysts under development include the heparosan synthase enzyme, pmHS, and its derivatives. Heparin-like polymers will interact with other proteins in the vertebrate body including coagulation factors and growth factor receptors. We expect that pmHS will possess certain catalytic properties exhibited by pmHAS and pmCS.

Conclusions Overall, depending on the choice of the catalyst, the acceptor molecule and the UDP-sugar precursors, a wide variety of potentially useful GAG-based materials based on the hyaluronan, chondroitin, and/or N-acetylheparosan may be created. These new syntheses allow unprecedented control of sugar chain length, size distribution, and composition.

Acknowledgements I thank Drs. Wei Jing and F. Michael Haller, Tasha A. Arnett, Bruce A. Baggenstoss, Daniel F. Gay, Leonard C. Oatman, Breca S. Tracy, and Carissa L. White for performing various experiments and laboratory support. This work


245 was supported in part by grants MCB-9876193 from National Science Foundation, OARS program AR02.2-019 from the Oklahoma Center for Advancement of Science and Technology, and GM56497 from National Institutes of Health, and a sponsored research agreement from Hyalose LLC.


References

1. Roberts, I.S. Annu. Rev. Microbiol. 1996, 50, 285-315. 2. DeAngelis, P.L. Glycobiology 2002, 12, 9R-16R. 3. DeAngelis, P.L.; Jing, W.; Drake, R.R.; Achyuthan, A.M. J. Biol. Chem. 1998, 273, 8454-8458. 4. DeAngelis, P.L.; Padgett-McCue, A.J. J. Biol. Chem. 2000, 275, 2412424129. 5. DeAngelis, P.L.; White, C.L. J. Biol. Chem. 2002, 277, 7209-7213. 6. DeAngelis, P.L. J. Biol. Chem. 1999, 274, 26557-26562. 7. DeAngelis, P.L. 2002 U.S. Patent 6,444,447. 8. Jing, W.; DeAngelis, P.L. Glycobiology. 2000, 10, 883-889. 9. Jing, W; DeAngelis, P.L. Glycobiology 2003, 13, 661-671. 10. DeAngelis, P.L.; Oatman, L.C.; Gay, D.F. J. Biol. Chem. 2003, 278, 3519935203. 11. Lee, H.G.; Cowman, M.K. Anal. Biochem. 1994, 219, 278-287. 12. Jing, W.; DeAngelis, P.L. unpublished. 13. Toole, B.P.; Wight, T.N.; Tammi, M.I. J. Biol. Chem. 2002, 277, 45934596. 14. Tracy, B.S.; DeAngelis, P.L. unpublished 15. DeAngelis, P.L.; Gay, D.F. unpublished


Sugar Polymer Engineering with Glycosaminoglycan Synthase Enzymes

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