Synchronized Chemoenzymatic Synthesis of Monodisperse Hyaluronan Polymers

Abstract

The length of the hyaluronan (HA) polysaccharide chain dictates its biological effects in many cellular and tissue systems. Long and short HA polymers often appear to have antagonistic or inverse effects. However, no source of very defined, uniform HA polymers with sizes greater than 10 kDa is currently available. We present a method to produce synthetic HA with very narrow size distributions in the range of ∼16 kDa to ∼2 MDa. The Pasteurella HA synthase enzyme, pmHAS, catalyzes the synthesis of HA polymer utilizing monosaccharides from UDP-sugar precursors. Recombinant pmHAS will also elongate exogenously supplied HA oligosaccharide acceptors in vitro in a nonprocessive fashion. As a result of bypassing the slow initiation step in vitro, the elongation process is synchronized in the presence of acceptor; thus all of polymer products are very similar in length. In contrast, without the use of an acceptor, the final polymer size range is difficult to predict and the products are more polydisperse. HA polymers of a desired size are constructed by controlling the reaction stoichiometry (i.e. molar ratio of precursors and acceptor molecules). The use of modified acceptors allows the synthesis of HA polymers containing tags (e.g. fluorescent, radioactive). In this scheme, each molecule has a single foreign moiety at the reducing terminus. Alternatively, the use of radioactive UDP-sugar precursors allows the synthesis of uniformly labeled native HA polymers. Overall, synthetic HA reagents with monodisperse size distributions and defined structures should assist in the elucidation of the numerous roles of HA in health and disease. The length of the hyaluronan (HA) polysaccharide chain dictates its biological effects in many cellular and tissue systems. Long and short HA polymers often appear to have antagonistic or inverse effects. However, no source of very defined, uniform HA polymers with sizes greater than 10 kDa is currently available. We present a method to produce synthetic HA with very narrow size distributions in the range of ∼16 kDa to ∼2 MDa. The Pasteurella HA synthase enzyme, pmHAS, catalyzes the synthesis of HA polymer utilizing monosaccharides from UDP-sugar precursors. Recombinant pmHAS will also elongate exogenously supplied HA oligosaccharide acceptors in vitro in a nonprocessive fashion. As a result of bypassing the slow initiation step in vitro, the elongation process is synchronized in the presence of acceptor; thus all of polymer products are very similar in length. In contrast, without the use of an acceptor, the final polymer size range is difficult to predict and the products are more polydisperse. HA polymers of a desired size are constructed by controlling the reaction stoichiometry (i.e. molar ratio of precursors and acceptor molecules). The use of modified acceptors allows the synthesis of HA polymers containing tags (e.g. fluorescent, radioactive). In this scheme, each molecule has a single foreign moiety at the reducing terminus. Alternatively, the use of radioactive UDP-sugar precursors allows the synthesis of uniformly labeled native HA polymers. Overall, synthetic HA reagents with monodisperse size distributions and defined structures should assist in the elucidation of the numerous roles of HA in health and disease. Hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan or hyaluronic acid; pmHAS, Pasteurella multocida HA synthase; GlcUA, glucuronic acid; SEC-MALLS, size exclusion chromatography multi-angle laser light scattering; Mn, number average molecular mass; Mw, weight average molecular mass; MDa, million Daltons. is a polysaccharide chain composed of repeating β4GlcUA-β3GlcNAc disaccharide units with molecular masses generally ranging from ∼104 to 107 Da in vertebrates and bacteria (1Toole B.P. Semin. Cell Dev. Biol. 2001; 12: 79-87Crossref PubMed Scopus (442) Google Scholar, 2Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (453) Google Scholar, 3Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar, 4Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (601) Google Scholar, 5DeAngelis P.L. Glycobiology. 2002; 12: 9R-16RCrossref PubMed Google Scholar). In animals, HA plays structural, recognition, and signaling roles. Certain pathogenic bacteria, namely Streptococcus Groups A and C and Pasteurella multocida Type A, utilize extracellular HA polysaccharide capsules to avoid host defenses and to increase virulence. The biological functions and biomedical applications of HA have long been of interest. It is now recognized that the HA of different sizes can have dramatically different effects on cellular behavior and growth (6Fieber C. Baumann P. Vallon R. Termeer C. Simon J.C. Hofmann M. Angel P. Herrlich P. Sleeman J.P. J. Cell Sci. 2004; 117: 359-367Crossref PubMed Scopus (142) Google Scholar, 7Termeer C.C. Hennies J. Voith U. Ahrens T. Weiss J.M. Prehm P. Simon J.C. J. Immunol. 2000; 165: 1863-1870Crossref PubMed Scopus (334) Google Scholar, 8McKee C.M. Penno M.B. Cowman M. Burdick M.D. Strieter R.M. Bao C. Noble P.W. J. Clin. Investig. 1996; 98: 2403-2413Crossref PubMed Scopus (700) Google Scholar, 9Toole B.P. Glycobiology. 2002; 12: 37R-42RCrossref PubMed Scopus (183) Google Scholar, 10West D.C. Kumar S. CIBA Found. Symp. 1989; 143: 187-207PubMed Google Scholar). Vertebrates may be able to control HA size in vivo by differential expression of biosynthetic enzymes (11Itano N. Kimata K. IUBMB Life. 2002; 54: 195-199Crossref PubMed Scopus (312) Google Scholar, 12Spicer A.P. Kaback L.A. Smith T.J. Seldin M.F. J. Biol. Chem. 1998; 273: 25117-25124Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Currently, the major method to generate HA is extraction from either rooster (chicken) comb or bacterial cultures. These HA preparations not only contain potential components from the original sources that could cause immunogenic, inflammatory, or allergenic responses but are also mixtures of a wide range of molecular masses. To interpret accurately the various biological functions of HA and to synthesize better HA-containing biomedical products, it is necessary to obtain uniform size-defined HA. HA synthases are the enzymes from vertebrates and microbes that polymerize the HA chain using UDP-sugar nucleotide precursors. The Class II HA synthase, pmHAS from Gram-negative P. multocida Type A, uses two separate glyco-syltransferase sites to add GlcNAc and GlcUA monosaccharides to the nascent HA chain (13Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar, 14Jing W. DeAngelis P.L. Glycobiology. 2003; 13: 661-671Crossref PubMed Scopus (65) Google Scholar, 15DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). A useful property of pmHAS is that it can extend exogenously provided HA acceptor oligosaccharides in vitro (16DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This polymerization reaction occurs in a nonprocessive fashion where the enzyme binds, elongates, and releases the growing HA chain in a rapid repetitive fashion (13Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar, 17DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In this report, we utilize the acceptor elongation activity for the chemoenzymatic synthesis of a variety of HA polymers with narrow size distributions. Acceptor Preparation—All of the reagents were the highest grade available from either Sigma or Fisher unless otherwise noted. The tetrasaccharide HA4, the starting acceptor for the synthesis of longer polymers, was generated by exhaustive degradation of streptococcal HA polymer with ovine testicular hyaluronidase Type V and purified by extensive chloroform extraction, ultrafiltration, and size exclusion chromatography on P2 (Bio-Rad) resin with 0.2 m ammonium formate buffer. The volatile salts were removed by repeated lyophilization from water. The HA4 molecule was converted into a fluorescent derivative in two steps. First, an amino-HA4 derivative was prepared by reductive amination of HA4 (12 mm) with sodium cyanoborohydride (70 mm) and excess diaminoethane (200 mm) in 0.1 m borate buffer, pH 8.5, 1 mm CuCl2 at 37 °C for 2 days. The amino-HA4 product was purified on P2 resin. Second, a fluorescent acceptor was prepared by derivatizing amino-HA4 with the N-hydroxysuccinimide ester of Oregon Green™ 488 (3-fold molar excess; Molecular Probes, Eugene, OR) in 50% dimethyl sulfoxide, 100 mm Hepes buffer, pH 8.5. The major isomer of fluor-HA4 was purified by preparative normal-phase thin layer chromatography (2:1:1 n-butanol/acetic acid/water and silica, Whatman). The identities of HA4, amino-HA4, and fluor-HA4 were verified by virtue of the agreement of their expected and experimental masses (775, 819, and 1213 Da, respectively) as assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in negative mode (17DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Catalyst Preparation and in Vitro Synthesis—The catalyst, pmHAS1–703, is a soluble purified Escherichia coli derived recombinant protein (13Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar). The enzyme in the octyl thioglucoside cell extracts was purified by chromatography on Toyopearl Red AF resin (Tosoh) using salt elution (50 mm Hepes, pH 7.2, 1 m ethylene glycol (an enzyme stabilizer) with 0–1.5 m NaCl gradient in 1 h) (17DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The fractions containing the target protein (≥90% pure by SDS-PAGE/Coomassie Blue-staining) were concentrated and exchanged into 1 m ethylene glycol, 50 mm Tris, pH 7.2, by ultrafiltration with an Amicon spin unit (Millipore). The syntheses in general contained pmHAS1–703, UDP-GlcNAc, UDP-GlcUA, 5 mm MnCl2,1 m ethylene glycol, and 50 mm Tris, pH 7.2. Reactions were incubated at 30 °C for 2–72 h. Analysis of in Vitro Synthesized HA—The size of HA was analyzed on agarose gels (0.7–1.2%; 1× TAE buffer (40 mm Tris acetate, 2 mm EDTA); 40 V) stained with Stains-All dye (0.005% w/v in ethanol) (18Lee H.G. Cowman M.K. Anal. Biochem. 1994; 219: 278-287Crossref PubMed Scopus (261) Google Scholar). Approximately 0.5–5 μg of HA was loaded per lane. For smaller HA polymers (<40 kDa), HA was also analyzed on polyacrylamide gels (15–20%) with acridine orange staining (19Ikegami-Kawai M. Takahashi T. Anal. Biochem. 2002; 311: 157-165Crossref PubMed Scopus (45) Google Scholar). To purify HA for later analysis, pmHAS was removed by chloroform extraction and the HA product was precipitated with three volumes of ethanol and the pellets were redissolved in water. Alternatively, the unincorporated precursor sugars were removed by ultrafiltration (Microcon units, Millipore). The HA concentration was determined by the carbazole assay using a glucuronic acid standard (20Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5218) Google Scholar). Size-exclusion chromatography/multi-angle laser light scattering (SEC-MALLS) analysis was employed to determine the absolute molecular masses of HA products. Polymers (2.5–12 μg mass; 50-μl injection) were separated on PL Aquagel-OH 30 (8 μm), -OH 40, -OH 50, -OH 60 (15 μm) columns (7.5 × 300 mm, Polymer Laboratories, Amherst, MA) in tandem or alone as required by the size range of the polymers to be analyzed. The columns were eluted with 50 mm sodium phosphate, 150 mm NaCl, pH 7, at 0.5 ml/min. MALLS analysis of the eluant was performed by a DAWN DSP Laser Photometer in series with an OPTI-LAB DSP interferometric refractometer (632.8 nm; Wyatt Technology, Santa Barbara, CA). The ASTRA software package was used to determine the absolute average molecular mass using a dn/dc coefficient of 0.153 determined by Wyatt Technology. The Mw and polydispersity values are the average of data from at least two SEC-MALLS runs. Acceptor-mediated Synchronization of Reaction Yields Monodisperse HA Products—Recombinant pmHAS synthesizes HA chains in vitro if supplied with both required UDP-sugars (15DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) according to Equation 1nUDP-GlcUA+nUDP-GlcNAc→2nUDP + [GlcUA-GlcNAc]neq.1 However, if a HA-like oligosaccharide ([GlcUA-GlcNAc]x) is also supplied in vitro, the overall incorporation rate was elevated up to ∼50–100-fold (16DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We suggested that the rate of initiation of a new HA chain de novo was slower than the subsequent elongation (i.e. repetitive addition of sugars to a nascent HA molecule). The observed stimulation of synthesis by exogenous acceptor appears to operate by bypassing the kinetically slower initiation step, allowing the elongation reaction to predominate as shown in Equation 2nUDP-GlcU A + nUDP-GlcNAc +[GlcUA-GlcNAc]x→2nUDP + [GlcUA-GlcNAc]x+neq.2 We performed HA polymerization reactions with purified pmHAS and UDP-sugar precursors under various conditions and analyzed the reaction products by agarose gel electrophoresis and/or size exclusion chromatography with MALLS. We observed that the size distribution of HA products obtained was quite different depending on the presence or the absence of the HA4 acceptor. In summary, the reactions with acceptor produced smaller HA chains with a more narrow size distribution. An example is depicted in Figs. 1 and 2 where the reaction containing HA4 acceptor yielded a HA product with a Mw of 555 kDa and polydispersity (Mw/Mn; Mn = number average molecular weight) of 1.006, but the parallel reaction without acceptor resulted in a product with a Mw of 1.8 MDa and Mw/Mn of 1.17. For reference, the polydispersity value for an ideal monodisperse polymer equals 1.Fig. 2SEC-MALLS analysis of in vitro HA synthesis products in the presence or absence of HA4 acceptor. The refractometer concentration peaks (lines) and the molar mass curves (symbols with corresponding y axis scale) of the matched set of reactions described in Fig. 1 are shown on the same PL Aquagel-OH 60 SEC column profile. A smaller narrow size distribution HA polymer is formed by pmHAS in the presence of HA4 (thick line and squares) as evidenced by its later elution time and flatter molar mass curve in comparison to the reaction without acceptor (thin line and circles).View Large Image Figure ViewerDownload (PPT) To explain the findings above, we further hypothesize that polymerization by pmHAS in the presence of an HA acceptor is a synchronized process. Reactions without the acceptor exhibit a lag period interspersed with numerous out of step initiation events that yield a short HA oligosaccharide (Fig. 3A). Once any HA chain is formed, the polymer is elongated rapidly. Other new HA chains that arise later during the lag period are also elongated rapidly, but the size of these younger chains never catches up to the older chains in a reaction with a finite amount of UDP-sugars. In contrast, in reactions containing an acceptor, all of the HA chains are elongated in parallel in a nonprocessive fashion resulting in a more homogenous final polymer population (Fig. 3B). For practical synthesis where there are more acceptor molecules than catalyst molecules, it is critical that processive elongation (i.e. no dissociation of the nascent HA chain and the synthase until polymerization is complete) does not occur because disparity would arise when some acceptor chains are elongated before other chains. Stoichiometric Control of HA Product Size—The two enzymological properties of recombinant pmHAS described above also allow for the control of HA polymer size in chemoenzymatic syntheses. First, as noted above, the rate-limiting step in vitro appears to be chain initiation. Therefore, pmHAS will transfer monosaccharides onto the existing HA acceptor chains before substantial de novo synthesis. Second, the enzyme polymerizes HA in a rapid nonprocessive fashion in vitro (13Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (97) Google Scholar, 17DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Therefore, the amount of HA4 should affect the final size of the HA product when a finite amount of UDP-sugar is present. The synthase will add all of the available UDP-sugar precursors to the nonreducing termini of acceptors as in the Equation 3nUDP−GlcUA+nUDP−GlcNAc +z[GlcUA−GlcNAc]x→2nUDP+z[GlcUA−GlcNAc]x+(n/z)eq.3 If there are many termini (i.e. z is large), a limited amount of UDP-sugars will be distributed among many molecules and thus result in many short polymer chain (Fig. if there are termini (i.e. z is the limited amount of UDP-sugars will be distributed among molecules and thus result in long polymer chain (Fig. 3B). To we performed a series of utilizing various of HA4 with a amount of UDP-sugar and pmHAS (Fig. this general we were able to generate HA from kDa to 2 MDa with polydispersity ranging from to (Fig. controlling the molar ratio of acceptor to it is now to the final HA polymer size of the starting UDP-sugars are in the reactions on the of HA polysaccharide of synthetic HA HA A variety of HA either by synchronized chemoenzymatic reactions in vitro or derived from streptococcal bacteria or sources were analyzed on a agarose gel with Stains-All The Mw of each synthetic HA polymer was determined by a of synthetic HA polymers produced in different reactions to and a of synthetic HA polymers produced in different reactions to and synthetic rooster comb HA streptococcal HA and 7, streptococcal HA The of the synthetic HA polymers their in comparison to Large Image Figure ViewerDownload (PPT) if an molecular mass HA chain is prepared by this the chain may be elongated by more UDP-sugars to the reaction provided that the catalyst is present. The resulting polymers as on gels and appear quite monodisperse the reaction time of UDP-sugars. The resulting with molecular masses that HA polymers than oligosaccharides kDa to may also be as starting for chain elongation by pmHAS not In Vitro of or for the of monodisperse polymers also allows the use of a modified acceptor to synthesize HA polymers containing various of foreign The pmHAS monosaccharides to the nonreducing of the acceptor chain (16DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (89) Google thus the of the reducing is available for reaction by numerous An example is shown using fluorescent HA4 acceptor to produce fluorescent monodisperse HA of various sizes (Fig. radioactive or (e.g. HA polymer are with the modified acceptor not Alternatively, the of all or a of the UDP-sugars in a chemoenzymatic synthesis reaction with a radioactive precursor (e.g. is a very useful method to produce labeled HA not The of this method is that the radioactive HA does not contain any foreign that with biological or cause of HA—The molecular masses of available HA preparations are in the Da range T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar). For smaller HA polymers, degradation (e.g. hyaluronidase or (e.g. or or (e.g. are However, this process is not because it is the final yield of the HA size is and at least step is We can generate HA as as kDa with polydispersity with the synchronized synthesis If the synthesis of smaller monodisperse HA oligosaccharides in length or is it is to utilize a of with pmHAS enzymes GlcUA and a GlcNAc in an repetitive fashion (17DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). molecular weight HA preparations are available from or bacterial but and size distributions of available HA polymers are than there a substantial for uniform HA in biomedical J.M. Curr. Opin. 1999; PubMed Scopus Google Scholar). we have shown that narrow size molecular mass HA is also prepared by reactions (Fig. in on this product size range To determine the average molecular mass of HA, MALLS is the many to the molecular but the required The of HA on agarose gels with (18Lee H.G. Cowman M.K. Anal. Biochem. 1994; 219: 278-287Crossref PubMed Scopus (261) Google Scholar) is often used for this of this method the The original HA were not uniform or and the of HA and on agarose gels with of the agarose A of an of synthetic HA polymers with defined, narrow size distributions (Fig. an series of for the size of HA in experimental In allows the of a variety of synthetic HA with narrow size distributions and defined for the numerous roles of HA in health and disease. We also that similar reactions utilizing the other Pasteurella synthases P.L. Glycobiology. 2002; 12: 9R-16RCrossref PubMed Google Scholar) will allow the chemoenzymatic synthesis of monodisperse and polymers in the We C. Oatman for pmHAS for SEC-MALLS on the of and Molecular and for mass at the to Laser and and for