US20090197308A1 - Enzymatic synthesis of sulfated polysaccharides - Google Patents

Enzymatic synthesis of sulfated polysaccharides Download PDF

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US20090197308A1
US20090197308A1 US11/920,319 US92031906A US2009197308A1 US 20090197308 A1 US20090197308 A1 US 20090197308A1 US 92031906 A US92031906 A US 92031906A US 2009197308 A1 US2009197308 A1 US 2009197308A1
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ost
enzyme
polysaccharide
paps
substrate
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Jian Liu
Robert J. Linhardt
Fikri Y. Avci
Eva M. Munoz
Jinghua Chen
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University of North Carolina at Chapel Hill
Rensselaer Polytechnic Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/64—Preparation of S-glycosides, e.g. lincomycin

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  • the presently disclosed subject matter relates generally to methods of sulfating polysaccharides.
  • the presently disclosed subject matter relates to methods of sulfating polysaccharides using O-sulfotransferases, which includes a reaction condition that reduces potential inhibitory effects from sulfur donor byproducts.
  • Heparan sulfate is a ubiquitous component of the cell surface and extracellular matrix. It regulates a wide range of physiologic and pathophysiologic functions, including embryonic development and blood coagulation, and can facilitate viral infection (Esko and Selleck (2002) Annu. Rev. Biochem. 71, 435-471; Liu and Thorp (2002) Med. Res. Rev. 22, 1-25). HS exerts its biological effects by interacting with the specific proteins involved in a given process (Capila and Lindhardt (2002) Angew. Chem. Int. Ed. 41, 390-412).
  • HS is a highly charged polysaccharide consisting of 1 ⁇ 4-linked glucosamine and glucuronic/iduronic acid units that contain both N- and O-sulfo groups. Unique saccharide sequences within HS determine the specificity of the binding of HS to its target proteins (Linhardt (2003) J. Med. Chem. 46, 2551-2564). Heparin, a specialized form of HS, is a commonly used anticoagulant drug. Thus, new methods for the synthesis of heparin and HS attract considerable interest for those developing anticoagulant and other HS-related drugs having improved pharmacological effects.
  • HS oligosaccharides Chemical synthesis has been the major route to obtain structurally defined heparin and HS oligosaccharides (Petitou and van Boeckel (2004) Angew. Chem. Int. Ed. 43, 3118-3133).
  • One example of a chemically synthesized HS oligosaccharide is a synthetic pentasaccharide having antithrombin-binding properties marketed in the United States under the trade name ARIXTRA® (GlaxoSmithKline, Middlesex, United Kingdom).
  • ARIXTRA® is a specific factor Xa inhibitor that is used clinically to prevent venous thromboembolic incidents during surgery.
  • HS is initially synthesized as a copolymer of D-glucuronic acid and N-acetylglucosamine (GlcNAc) through the action of D-glucuronyl and N-acetyl-D-glucosaminyltransferase (Lindahl et al. (1998) J. Biol. Chem. 273, 24979-24982).
  • GlcNAc N-acetylglucosamine
  • N-deacetylation and N-sulfation (carried out by N-deacetylase/N-sulfotransferase) of the glucosamine residue to form N-sulfoglucosamine (GlcNS), C 5 epimerization of glucuronic acid (carried out by epimerase) to form L-iduronic acid (IdoUA), 2-O-sulfation of IdoUA (carried out by 2-O-sulfotransferase (2-OST)), 6-O-sulfation of glucosamine (carried out by 6-O-sulfotransferase (6-OST)), and 3-O-sulfation of glucosamine (carried out by 3-O-sulfotransferase (3-OST)) (Sasisekharan et al. (2002) Nat. Rev. Cancer 2, 521-528).
  • 2-OST 2-O-sulfation of IdoUA
  • Enzymes “in the pathway” for HS biosynthesis have been cloned and expressed, and have been employed in the synthesis of HS polysaccharides.
  • Kuberan and Rosenberg (Balagurunathan et al. (2003) Nat. Biotechnol. 21, 1343-1346; Kuberan et al. (2003) J. Am. Chem. Soc. 125, 12424-12425; Balagurunathan et al. (2003) J. Biol. Chem. 278, 52613-52621) utilized these enzymes to synthesize an HS containing antithrombin binding sites with anticoagulant activity.
  • OST catalyzed sulfation reaction utilizes 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfur donor, producing adenosine 3′,5′-diphosphate (PAP).
  • PAPS 3′-phosphoadenosine 5′-phosphosulfate
  • PAP can compete with PAPS for OST binding, which can result in inhibition of the sulfation reaction over time as PAP concentration increases in the reaction mixture.
  • a method of sulfating a polysaccharide comprising (a) providing a reaction mixture comprising at least one O-sulfotransferase (OST) enzyme and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); (b) incubating a polysaccharide substrate with the reaction mixture, wherein production of the sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to adenosine 3′,5′-diphosphate (PAP); and (c) providing a reaction condition which modifies PAP to reduce an inhibitory effect of PAP on the polysaccharide sulfation.
  • OST O-sulfotransferase
  • PAPS 3′-phosphoadenosine 5′-phosphosulfate
  • providing the reaction condition comprises providing a PAPS regeneration system comprising a PAPS regenerating enzyme and a sulfur donor compound, wherein the PAPS regenerating enzyme catalyzes regeneration of the PAPS from the PAP utilizing the sulfur donor compound as a substrate.
  • the PAPS regenerating enzyme is an arylsulfotransferase, such as for example AST-IV.
  • the sulfur donor compound is an aryl sulfate compound, such as for example p-nitrophenol sulfate (PNPS).
  • providing the reaction condition comprises providing a phosphatase enzyme, wherein the phosphatase enzyme modifies the PAP.
  • a method of sulfating a polysaccharide comprising (a) providing a reaction mixture comprising PAP, a PAPS regenerating enzyme and a sulfur donor compound; (b) incubating the reaction mixture for a time period sufficient to catalyze the production of PAPS from the PAP by the PAPS regenerating enzyme utilizing the sulfur donor compound as a substrate, such as for example a time period from about 1 minute to about 30 minutes; and (c) incubating a polysaccharide substrate and at least one OST enzyme with the reaction mixture, wherein production of a sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to PAP and wherein the PAPS regenerating enzyme catalyzes regeneration of the PAPS from the PAP utilizing the sulfur donor compound as a substrate.
  • the PAPS regenerating enzyme is an arylsulfotransferase, such as for example AST-IV.
  • the donor compound is an aryl sulfate compound, such as for example PNPS.
  • the at least one OST enzyme is selected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST, and combinations thereof.
  • the at least one OST enzyme is a recombinant OST enzyme, which is, in some embodiments, produced in a bacterial expression system.
  • the OST enzyme is a fusion protein, such as for example a maltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OST fusion protein.
  • the OST enzyme is immobilized on a substrate, such as for example an agarose bead.
  • the polysaccharide substrate is a chemically desulfated N-sulfated (CDSNS) heparin.
  • CDSNS chemically desulfated N-sulfated
  • the polysaccharide substrate is partially sulfated prior to reaction mixture incubation.
  • the sulfated polysaccharide is a glycosaminoglycan (GAG), such as for example a heparan sulfate (HS).
  • the sulfated polysaccharide is an HS that is an anticoagulant-active HS, an antithrombin-binding HS, a fibroblast growth factor (FGF)-binding HS, a herpes simplex virus envelope glycoprotein D-binding HS, or has a combination of these properties.
  • an anticoagulant-active HS an antithrombin-binding HS, a fibroblast growth factor (FGF)-binding HS, a herpes simplex virus envelope glycoprotein D-binding HS, or has a combination of these properties.
  • FGF fibroblast growth factor
  • kits for sulfating a polysaccharide comprises at least one OST enzyme and a reagent which modifies PAP to reduce an inhibitory effect of PAP on polysaccharide sulfation.
  • the kit further comprising instructions for sulfating a polysaccharide.
  • the at least one OST enzyme is contained within a first container and the reagent is contained within a second container.
  • the at least one OST enzyme is selected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST, and combinations thereof.
  • the OST enzyme is a recombinant OST enzyme, such as for example a recombinant OST enzyme produced in a bacterial expression system.
  • the OST enzyme is a fusion protein, such as for example a maltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OST fusion protein.
  • the OST enzyme is immobilized on a substrate, such as for example an agarose bead.
  • the reagent comprises a PAPS regeneration system comprising a PAPS regenerating enzyme (e.g., an arylsulfotransferase, such as AST-IV) and a sulfur donor compound (e.g., an aryl sulfate compound, such as PNPS.
  • a PAPS regenerating enzyme e.g., an arylsulfotransferase, such as AST-IV
  • a sulfur donor compound e.g., an aryl sulfate compound, such as PNPS.
  • the reagent comprises a phosphatase enzyme.
  • FIGS. 1A and 1B are schematic drawings showing synthesis of sulfated polysaccharides coupled with a PAPS enzymatic regeneration system.
  • FIG. 1A shows the stepwise enzymatic synthesis of sulfated polysaccharides using HS O-sulfotransferases. The description of intermediate polysaccharides is disclosed in the Examples herein below.
  • Compounds 4a and 4b were prepared by inverting the order of sulfation steps. 4a was prepared by incubating compound 1 with 2-OST followed by 6-OST, whereas 4b was prepared by incubating compound 1 with 6-OST followed by 2-OST.
  • FIG. 1B shows the reaction catalyzed by arylsulfotransferase IV (AST-IV) to generate PAPS.
  • R represents —H or —SO 3 .
  • FIG. 2 is a graph showing activities of recycled immobilized sulfotransferases.
  • the immobilized enzymes were utilized in multiple cycles. The activities of the immobilized enzymes after each cycle were determined as described in the Examples herein below. A total of 10 cycles was conducted. 2-OST (•); 6-OST ( ⁇ ); 3-OST-1 ( ⁇ ); and 3-OST-3 ( ⁇ ).
  • FIGS. 3A-3E are RPIP-HPLC chromatograms of the disaccharide analysis of synthesized polysaccharides.
  • the synthesized polysaccharides were digested with a mixture of heparinases, including heparin lyase I, II, III, and heparinase IV.
  • the resultant disaccharides were purified by BioGel P-2 and resolved on RPIP-HPLC.
  • FIGS. 3A-3E show the chromatograms of the disaccharide analysis of compounds 1, 2, 3, 4a, and 4b, from FIG. 1A respectively.
  • FIGS. 4A and 4B are graphs showing the effect of the synthesized polysaccharides on FGF-2-dependent BaF3 FGFR1c cell proliferation.
  • FIG. 4A BaF3 FGFR1c cells were seeded in 96-well plates as described with 2 nM FGF2 for control and 2 nM FGF2 plus a 1 ⁇ g/ml concentration of the following compounds: heparin, 1, 2, 3, 4a, and 4b ( FIG. 1A ).
  • FIG. 4B shows dose-response curves of heparin, 4a, and 4b for their activities in stimulating cell proliferation. Cells were cultured for 40 hours (h), followed by incubating in the media containing [ 3 H]thymidine for 4 h. The cellular proliferation was determined by [ 3 H]thymidine incorporation into the DNA.
  • Heparan sulfates are highly sulfated polysaccharides, present on the surface of mammalian cells and in the extracellular matrix in large quantities.
  • HS is a highly charged polysaccharide consisting of 1 ⁇ 4-linked glucosamine and glucuronic/iduronic acid units that contain both N- and O-sulfo groups.
  • Heparin a specialized form of HS, is a commonly used anticoagulant drug.
  • HSs play critical roles in a variety of important biological processes, including assisting viral infection, regulating blood coagulation and embryonic development, suppressing tumor growth, and controlling the eating behavior of test subjects by interacting with specific regulatory proteins (Liu, J., and Thorp, S. C. (2002) Med. Res. Rev. 22:1-25; Rosenberg, R. D., et al., (1997) J. Clin. Invest. 99:2062-2070; Bernfield, M., et al., (1999) Annu. Rev. Biochem. 68:729-777; Alexander, C. M., et al., (2000) Nat. Genet. 25:329-332; Reizes, O., et al., (2001) Cell 106:105-116).
  • the unique sequences determine to which specific proteins HSs bind, thereby regulating biological processes.
  • HS The biosynthesis of HS occurs in the Golgi apparatus. It is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by D-glucuronyl and N-acetyl-D-glucosaminyltransferase, followed by various modifications (Lindahl, U., et al., (1998) J. Biol. Chem. 273:24979-24982).
  • HS N-deacetylase/N-sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotransferase are present in multiple isoforms.
  • Each isoform is believed to recognize a saccharide sequence around the modification site in order to generate a specific sulfated saccharide sequence (Liu, J., et al., (1999) J. Biol. Chem. 274:5185-5192; Aikawa, J.-I., et al., (2001) J. Biol. Chem.
  • HS D-glucosaminyl 3-O-sulfotransferase (3-OST) isoforms generate 3-O-sulfated glucosamine residues that are linked to different sulfated uronic acid residues.
  • 3-OST isoform 1 (3-OST-1) transfers sulfate to the 3-OH position of an N-sulfated glucosamine residue that is linked to a glucuronic acid residue at the nonreducing end (GlcUA-GlcNS ⁇ 6S).
  • 3-OST isoform 3 (3-OST-3) transfers sulfate to the 3-OH position of an N-unsubstituted glucosamine residue that is linked to a 2-O-sulfated iduronic acid at the nonreducing end (IdoUA2S-GlcNH 2 ⁇ 6S) (Liu, J., et al., (1999) J. Biol. Chem. 274:38155-38162).
  • the difference in the substrate specificity of 3-OSTs results in distinct biological functions.
  • the HS modified by 3-OST-1 binds to antithrombin (AT) and possesses anticoagulant activity (Liu, J., et al., (1996) J. Biol. Chem.
  • HS modified by 3-OST-3 (3-OST-3A and 3-OST-3B) binds to glycoprotein D (gD) of herpes simplex virus, type 1, (HSV-1) thus mediating viral entry (Shukla, D., et al., (1999) Cell 99:13-22).
  • gD glycoprotein D
  • HSV-1 herpes simplex virus
  • HS- and heparin-regulated anticoagulation mechanisms have been studied extensively. It is now known that HS, including heparin, interact with AT, a serine protease inhibitor, to inhibit the activities of thrombin and factor Xa in the blood coagulation cascade (Rosenberg, R. D., et al., (1997) J. Clin. Invest 99:2062-2070). Anticoagulant-active HS(HS act ) and heparin contain one or multiple AT-binding sites per polysaccharide chain.
  • This binding site contains a specific pentasaccharide sequence with a structure of -GlcNS(or Ac)6S-GlcUA-GlcNS3S( ⁇ 6S)-IdoUA2S-GlcNS6S-.
  • the 3-O-sulfation of glucosamine for generating GlcNS3S( ⁇ 6S) residue, which is carried out by 3-OST-1, is an important modification for the synthesis of HS act (Liu, J., et al., (1996) J. Biol. Chem. 271:27072-27082; Shworak, N. W., et al., (1997) J. Biol. Chem. 272:28008-28019).
  • the presently disclosed subject matter provides enzymatic methods for the sulfation of multimilligram amounts of heparan sulfate having particular functions using sulfotransferases coupled with a system for reducing inhibitory effects from sulfur donor byproducts.
  • the system for reducing inhibitory byproducts comprises a 3′-phosphoadenosine 5′-phosphosulfate regeneration system.
  • the system comprises a phosphatase enzyme.
  • the presently disclosed subject matter employs recombinant sulfotransferases. Because the recombinant sulfotransferases can be recombinantly expressed in bacteria, and the disclosed methods can use low cost sulfo donors, the presently disclosed subject matter can be readily utilized to synthesize large quantities of biologically active heparan sulfates while reducing the production of reaction inhibitory byproducts.
  • PAP adenosine 3′,5′-diphosphate
  • a PAPS regeneration system such as the system developed by Burkhart and colleagues (Burkart et al. (2000) J. Org. Chem. 65, 5565-5574, incorporated herein by reference), has been modified and adapted to be coupled to the enzymatic synthesis reactions.
  • the PAPS regeneration system converts PAP into PAPS, thereby reducing accumulation of inhibitory PAP in the reaction mixture and reducing production costs related to providing PAPS to drive the sulfation reaction.
  • phosphatase enzymes can be utilized to modify PAP so that it no longer has binding affinity for sulfotransferases.
  • the presently disclosed sulfation system can be adapted to produce a multitude of HS molecules having varied biological activities by selecting appropriate sulfotransferases to include and by sequentially controlling the addition of those sulfotransferases to the reaction system to facilitate appropriate timing of sulfations of the polysaccharide template.
  • HS having specific biological activities can be synthesized utilizing the presently disclosed methods, including anticoagulant HS, fibroblast growth factor-2-binding activity, herpes simplex virus glycoprotein D (gD)-binding HS, and fibroblast growth factor 2 (FGF2) receptor-binding HS.
  • FIG. 1A Only two or three enzymatic steps are required for the synthesis of each of these biologically-active HS molecules ( FIG. 1A ).
  • the presently disclosed subject matter provides an efficient and effective method for the large scale synthesis of a wide range of HS with specific activity required.
  • this method provides a model system to better understand the biosynthesis of HS.
  • a method of sulfating a polysaccharide comprises incubating a polysaccharide substrate to be sulfated with a reaction mixture that comprises at least one sulfotransferase enzyme, such as for example an O-sulfotransferase (OST) enzyme, and a sulfur donor, such as for example PAPS.
  • a reaction mixture that comprises at least one sulfotransferase enzyme, such as for example an O-sulfotransferase (OST) enzyme, and a sulfur donor, such as for example PAPS.
  • OST O-sulfotransferase
  • PAPS sulfur donor
  • Production of the sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to adenosine 3′,5′-diphosphate (PAP).
  • PAP adenosine 3′,5′-diphosphate
  • a reaction condition is further provided that modifies generated PAP to reduce an inhibitory effect of PAP on the polysaccharide sulfation.
  • a PAPS regeneration system can be coupled with the sulfation reaction to convert PAP into PAPS or phosphatases can be added to the reaction mixture to modify PAP such that it does not compete with PAPS for binding with OSTs.
  • the polysaccharide substrate is a previously N,O-desulfated and re-N-sulfated polysaccharide, such as for example a chemically desulfated N-sulfated (CDSNS) heparin.
  • CDSNS chemically desulfated N-sulfated
  • the polysaccharide substrate is partially sulfated prior to reaction mixture incubation.
  • a CDSNS can be reacted with a particular OST to produce a sulfated polysaccharide intermediate product that can then be reacted with a different OST to further sulfate the polysaccharide at different locations.
  • FIG. 1A schematically illustrates several exemplary polysaccharide substrates sequentially reacted with different OSTs to produce different intermediate and end products.
  • compound 1 can be reacted with 2-OST and then 6-OST to produce compound 4a or compound 1 can be reacted with 6-OST and then 2-OST to produce compound 4b, each having fibroblast growth factor (FGF)-binding activity.
  • FGF fibroblast growth factor
  • compounds 4a or 4b can be reacted with 3-OST-1 to produce compound 5 having antithrombin-binding and anticoagulant activities.
  • compounds 4a or 4b can be reacted with 3-OST-3 to produce compound 6 having herpes simplex virus (HSV) envelope glycoprotein D (gD) binding activity.
  • HSV herpes simplex virus
  • gD envelope glycoprotein D
  • the presently disclosed subject matter provides for the production of HS compounds having different biological properties based on the selection and sequential reaction of different OSTs with polysaccharide substrates.
  • the polysaccharide substrate can be reacted with different OST enzymes by addition of each enzyme sequentially to the same reaction mixture, or intermediate polysaccharide products can be purified from the reaction mixture after reaction of a particular OST and then reacted with a different OST.
  • different OST enzymes can be added to the reaction mixture simultaneously.
  • the sulfated polysaccharide product can be a glycosaminoglycan (GAG).
  • GAGs are the most abundant heteropolysaccharides in the body. These molecules are long unbranched polysaccharides containing a repeating disaccharide unit. The disaccharide units can contain either of two modified sugars: N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) and a uronic acid such as glucuronate or iduronate.
  • GAGs are highly negatively charged molecules, with extended conformation that imparts high viscosity to the solution.
  • the sulfated polysaccharide product is a HS.
  • the sulfated polysaccharide product is an anticoagulant-active HS, an antithrombin-binding HS, an FGF-binding HS, and an HSV gD-binding HS.
  • sulfotransferases particularly O-sulfotransferases (OSTs)
  • OSTs O-sulfotransferases
  • Sulfotransferases comprise a family of enzymes that catalyze the transfer of a sulfonate or sulfuryl group (SO 3 ) from the cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to an acceptor molecule.
  • SO 3 sulfuryl group
  • PAPS cofactor 3′-phosphoadenosine-5′-phosphosulfate
  • the term sulfation is still widely used. Therefore, the term “sulfation” as used herein refers to a transfer of a sulfonate or sulfuryl group from one molecule to another.
  • Sulfotransferases mediate sulfation of different classes of substrates such as carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids for a variety of biological functions including signaling and modulation of receptor binding (Bowman et al., (1999) Chem. Biol. 6, R9-R22; and Falany (1997) FASEB J. 11, 1-2).
  • substrates such as carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids
  • Falany 1997 FASEB J. 11, 1-2.
  • Many new sulfotransferases have been identified and cloned (Aikawa et al., (1999) J. Biol. Chem. 274, 2690; Dooley (1998) Chemico - Biol. Interact. 109, 29; Fukuta et al. (1998) Biochim.
  • O-sulfotransferase includes polypeptides and nucleic acids encoding HS O-sulfotransferases, such as for example “2-OST” (e.g., mouse 2-OST, GENBANK® Accession No. AAC40135 (SEQ ID NO:1); “3-OST-1” (e.g., human 3-OST-1, GENBANK® Accession No. NP — 005105 (SEQ ID NO:2); “3-OST-3” (e.g., human 3-OST-3A, GENBANK® Accession No. NP — 006033 (SEQ ID NO:3) and human 3-OST-3B, GENBANK® Accession No.
  • 2-OST e.g., mouse 2-OST, GENBANK® Accession No. AAC40135 (SEQ ID NO:1)
  • 3-OST-1 e.g., human 3-OST-1, GENBANK® Accession No. NP — 005105 (SEQ ID NO:2)
  • NP — 006032 SEQ ID NO:4; and “6-OST” (e.g., mouse 6-OST-1, GENBANK® Accession No. NP — 056633 (SEQ ID NO:5), mouse 6-OST-2, GENBANK® Accession No. BAA89247 (SEQ ID NO:6), and mouse 6-OST-3, GENBANK® Accession No. NP — 056635 (SEQ ID NO:7)), which are HS 2-O-sulfotransferase, HS 3-O-sulfotransferase isoform 1, HS 3-O-sulfotransferase isoform 3, and HS 6-O-sulfotransferase, respectively.
  • 6-OST e.g., mouse 6-OST-1, GENBANK® Accession No. NP — 056633 (SEQ ID NO:5), mouse 6-OST-2, GENBANK® Accession No. BAA89247 (SEQ ID NO:6), and
  • OST includes invertebrate and vertebrate homologs of the O-sulfotransferases (e.g., mammalian (such as human and mouse), insect, and avian homologs).
  • O-sulfotransferases e.g., mammalian (such as human and mouse), insect, and avian homologs.
  • OST includes invertebrate and vertebrate homologs of the O-sulfotransferases (e.g., mammalian (such as human and mouse), insect, and avian homologs).
  • OST includes invertebrate and vertebrate homologs of the O-sulfotransferases (e.g., mammalian (such as human and mouse), insect, and avian homologs).
  • OST includes invertebrate and vertebrate homologs of the O-sulfotransferases (e.g., mammalian (such as human and mouse), insect, and
  • OST gene product refers to peptides having amino acid sequences which are substantially identical to native amino acid sequences from the organism of interest and which are biologically active in that they comprise all or a part of the amino acid sequence of a HS O-sulfotransferase isoform, or cross-react with antibodies raised against a HS O-sulfotransferase isoform polypeptide, or retain all or some of the biological activity of the native amino acid sequence or protein.
  • biological activity can include immunogenicity.
  • OST gene product also include analogs of HS O-sulfotransferase molecules.
  • analog is intended that a DNA or peptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some of the biological activity of those sequences. Analogs can be derived from genomic nucleotide sequences as are disclosed herein or from other organisms, or can be created synthetically. Those skilled in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or construct OST analogs.
  • OST gene product “OST protein”, and “OST polypeptide” to comprise all or substantially all of the amino acid sequence of a native OST gene product. Shorter or longer sequences are anticipated to be of use in the presently disclosed subject matter, shorter sequences are herein referred to as “segments.” Thus, the terms “OST gene product”, “OST protein”, and “OST polypeptide” also include fusion or recombinant HS O-sulfotransferase polypeptides and proteins comprising sequences of the OST protein. Methods of preparing such proteins are known in the art.
  • the OST is a 2-OST or a 6-OST fusion protein, such as a maltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OST fusion protein, as disclosed herein.
  • MBP maltose-binding protein
  • OST gene refers to any DNA sequence that is substantially identical to a polynucleotide sequence encoding a HS O-sulfotransferase isoform gene product, protein or polypeptide as defined above, and can also comprise any combination of associated control sequences.
  • the terms also refer to RNA, or antisense sequences, complementary to such DNA sequences.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
  • a DNA segment encoding a HS O-sulfotransferase polypeptide refers to a DNA segment that contains OST coding sequences, yet is isolated away from, or purified free from, total genomic DNA of a source species, such as for example Homo sapiens .
  • DNA segment includes DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.
  • substantially identical when used to define either a OST gene product or amino acid sequence, or a OST gene or nucleic acid sequence, means that a particular sequence varies from the sequence of a natural OST by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence.
  • sequences include “mutant” sequences, or sequences in which the biological activity is altered to some degree but retains at least some of the original biological activity.
  • DNA analog sequences are “substantially identical” to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions of the natural OST gene; or (b) the DNA analog sequence is capable of hybridization of DNA sequences of (a) under stringent conditions and which encode biologically active OST gene products; or (c) the DNA sequences are degenerate as a result of alternative genetic code to the DNA analog sequences defined in (a) and/or (b).
  • Substantially identical analog proteins will be greater than about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequence of the native protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
  • nucleic acid sequences In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequences or substitution of equivalent amino acids to create biologically functional equivalents.
  • Sequence identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group.
  • the GAP program utilizes the alignment method of Needleman et al. (1970) J Mol Biol 48:443, as revised by Smith et al. (1981) Adv Appl Math 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See Schwartz et al. (1979) Nuc Acids Res 6(2):745-755; Gribskov et al. (1986) Nuc Acids Res 14(1):327-334.
  • the present subject matter concerns the use of OST genes and gene products that include within their respective sequences a sequence that is essentially that of an OST gene, or the corresponding protein.
  • a sequence essentially as that of an OST gene means that the sequence is substantially identical or substantially similar to a portion of an OST gene and contains a minority of bases or amino acids (whether DNA or protein) which are not identical to those of an OST protein or an OST gene, or which are not a biologically functional equivalent.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein.
  • Nucleotide sequences are “essentially the same” where they have between about 75% and about 85% or more preferably, between about 86% and about 90%, or more preferably greater than 90%, or more preferably between about 91% and about 95%, or even more preferably, between about 96% and about 99%; of nucleic acid residues which are identical to the nucleotide sequence of a OST gene.
  • peptide sequences which have about 60%, 70%, 80%, or 90%, or preferably from 90-95%, or more preferably greater than 96%, or more preferably 95-98%, or most preferably 96%, 97%, 98%, or 99% amino acids which are identical or functionally equivalent or biologically functionally equivalent to the amino acids of an OST polypeptide will be sequences which are “essentially the same”.
  • OST gene products and OST encoding nucleic acid sequences which have functionally equivalent codons, are also covered by the presently disclosed subject matter.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the ACG and AGU codons for serine. Applicants contemplate substitution of functionally equivalent codons of Table 1 into sequences of OSTs disclosed herein as equivalents.
  • amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids or 5′ or 3′ nucleic acid sequences, and yet still be encompassed by the OSTs disclosed herein, so long as the sequence retains biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences which can, for example, include various non-coding sequences flanking either of the 5′ or 3′ portions of the coding region or can include various internal sequences, i.e., introns, which are known to occur within genes.
  • nucleotide segments that are complementary to the sequences of the present subject matter in one embodiment, segments that are fully complementary, i.e. complementary for their entire length.
  • Nucleic acid sequences that are “complementary” are those, which are base-paired according to the standard Watson-Crick complementarity rules.
  • complementary sequences means nucleic acid sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein.
  • a particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of about 30° C., typically in excess of about 37° C., and preferably in excess of about 45° C.
  • Stringent salt conditions will ordinarily be less than about 1,000 mM, typically less than about 500 mM, and preferably less than about 200 mM.
  • preferred conditions are salt concentration of about 200 mM and temperature of about 45° C.
  • One example of such stringent conditions is hybridization at 4 ⁇ SSC, at 65° C., followed by a washing in 0.1 ⁇ SSC at 65° C. for one hour.
  • Another exemplary stringent hybridization scheme uses 50% formamide, 4 ⁇ SSC at 42° C.
  • Another example of “stringent conditions” refers to conditions of high stringency, for example 6 ⁇ SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 ⁇ g/ml salmon sperm DNA and 15% formamide at 68° C.
  • Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50° C. and 10 ⁇ SSC (0.9 M NaCl/0.09 M sodium citrate) and remain bound when subjected to washing at 55° C. in 1 ⁇ SSC. Sequence identity can be determined by hybridization under stringent conditions, for example, at 50° C. or higher and 0.1 ⁇ SSC (9 mM NaCl/0.9 mM sodium citrate).
  • Nucleic acids that are substantially identical to the provided OSTs bind to the disclosed OSTs under stringent hybridization conditions.
  • probes particularly labeled probes of DNA sequences
  • the source of homologous genes can be any species, e.g., primate species, particularly human; rodents, such as rats and mice; canines; felines; bovines; ovines; equines; insects; yeasts; nematodes; etc.
  • homologs have substantial sequence similarity, i.e. at least 75% sequence identity between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which can be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nucleotides long, more usually at least about 30 nucleotides long, and can extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990) J Mol Biol 215:403-410. The sequences provided herein are essential for recognizing OST related and homologous proteins in database searches.
  • identity is just that, i.e. the same amino acid at the same relative position in a given family member of a gene family.
  • Homology and similarity are generally viewed as broader terms.
  • biochemically similar amino acids for example leucine and isoleucine or glutamate/aspartate, can be present at the same position—these are not identical per se, but are biochemically “similar”. As disclosed herein, these are referred to as conservative differences or conservative substitutions. This differs from a conservative mutation at the DNA level, which changes the nucleotide sequence without making a change in the encoded amino acid, e.g., TCC to TCA, both of which encode serine.
  • percent identities can be generated by alignments with the program GENEWORKSTM (Oxford Molecular, Inc. of Campbell, Calif., U.S.A.) and/or the BLAST program at the NCBI website. Another commonly used alignment program is entitled CLUSTAL W and is described in Thompson et al. (1994) Nucleic Acids Res 22(22):4673-4680, among other places.
  • gene is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences.
  • modifications and changes can be made in the structure of the OST proteins and peptides described herein and still constitute a molecule having like or otherwise desirable characteristics.
  • certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive capacity with, for example, structures in the nucleus of a cell. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or the nucleic acid sequence encoding it) to obtain a protein with the same, enhanced, or antagonistic properties.
  • Biologically functional equivalent peptides are peptides in which certain, but not most or all, of the amino acids can be substituted. Thus, applicants contemplate substitution of codons that encode biologically equivalent amino acids as described herein into the sequences of the disclosed OSTs, but which are not set forth herein in their entirety for convenience.
  • functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man can be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test OST mutants in order to examine OST sulfotransferase activity, or other activity at the molecular level.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.
  • arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are defined herein as biologically functional equivalents.
  • Other biologically functionally equivalent changes will be appreciated by those of skill in the art.
  • hydropathic index of amino acids can be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine ( ⁇ 0.4); threonine ( ⁇ 0.7); serine ( ⁇ 0.8); tryptophan ( ⁇ 0.9); tyrosine ( ⁇ 1.3); proline ( ⁇ 1.6); histidine ( ⁇ 3.2); glutamate ( ⁇ 3.5); glutamine ( ⁇ 3.5); aspartate ( ⁇ 3.5); asparagine ( ⁇ 3.5); lysine ( ⁇ 3.9); and arginine ( ⁇ 4.5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( ⁇ 0.4); proline ( ⁇ 0.5 ⁇ 1); alanine ( ⁇ 0.5); histidine ( ⁇ 0.5); cysteine ( ⁇ 1.0); methionine ( ⁇ 1.3); valine ( ⁇ 1.5); leucine ( ⁇ 1.8); isoleucine ( ⁇ 1.8); tyrosine ( ⁇ 2.3); phenylalanine ( ⁇ 2.5); tryptophan ( ⁇ 3.4).
  • Recombinant vectors and isolated DNA segments can therefore variously include the O-sulfotransferase polypeptide-encoding region itself, include coding regions bearing selected alterations or modifications in the basic coding region, or include larger polypeptides which nevertheless comprise the O-sulfotransferase polypeptide-encoding regions or can encode biologically functional equivalent proteins or peptides which have variant amino acid sequences.
  • Biological activity of an O-sulfotransferase can be determined using techniques generally known in the art, for example as disclosed herein in the Examples.
  • nucleic acid segments of the present subject matter can be combined with other DNA sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length can be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid fragments can be prepared which include a short stretch complementary to a nucleic acid sequence set forth in any of the OSTs disclosed herein, such as about 10 nucleotides, and which are up to 10,000 or 5,000 base pairs in length, with segments of 3,000 being preferred in certain cases. DNA segments with total lengths of about 4,000, 3,000, 2,000, 1,000, 500, 200, 100, and about 50 base pairs in length are also contemplated to be useful.
  • Recombinant vectors form further aspects of the present subject matter.
  • Particularly useful vectors are those in which the coding portion of the DNA segment is positioned under the control of a promoter.
  • the promoter can be that naturally associated with the OST gene, as can be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or polymerase chain reaction (PCR) technology and/or other methods known in the art, in conjunction with the compositions disclosed herein.
  • PCR polymerase chain reaction
  • a recombinant or heterologous promoter is a promoter that is not normally associated with a 3-O-sulfotransferase gene in its natural environment.
  • Such promoters can include promoters isolated from bacterial, viral, eukaryotic, or mammalian cells. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression.
  • promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology (See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual , Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • the promoters employed can be constitutive or inducible and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
  • the OST enzyme is immobilized on a substrate. This provides an advantage in that the substrate to which the OSTs are attached can be washed after a sulfation reaction to remove all components of the reaction except the bound OSTs. As such, the products of the reaction can be more easily separated from the enzymes catalyzing the reaction and the OSTs can be recycled and utilized again in multiple sulfation reactions.
  • the substrate is agarose.
  • the agarose substrate is an agarose bead and the OSTs are linked to the beads.
  • the presently disclosed method for sulfating polysaccharides can comprise providing a “reaction condition” that modifies PAP to reduce inhibitory effects of PAP, such as competing with PAPS for binding with OSTs, on the polysaccharide sulfation.
  • the reaction condition comprises a phosphatase enzyme.
  • the phosphatase enzyme can remove a phosphate from the PAP, which reduces its binding affinity for OSTs.
  • the phosphatase is 3′-ribonucleotide phosphohydrolase.
  • the reaction condition is a PAPS regeneration system, which comprises a PAPS regenerating enzyme and a sulfur donor compound.
  • the PAPS regenerating enzyme catalyzes regeneration of the PAPS from the PAP utilizing the sulfur donor compound as a substrate. See, e.g., U.S. Pat. No. 6,255,088; Burkart et al., (2000) J. Org. Chem. 65, 5565-5574, which is herein incorporated by reference in its entirety.
  • the PAPS regeneration system provides the dual advantages of reducing the inhibitory effects of PAP accumulation on sulfotransferase activity while also constantly “recharging” the reaction mixture with the primary sulfur donor molecule, PAPS.
  • an aspect of the presently disclosed subject matter is directed to a sulfur donor compound (e.g., PAPS) regeneration process coupled with sulfation of a polysaccharide substrate.
  • the process can be of a type wherein the sulfation of a polysaccharide substrate is catalyzed by a sulfotransferase, such as one or more OSTs, with a conversion of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to adenosine 3′,5′-diphosphate (PAP).
  • PAPS 3′-phosphoadenosine-5′-phosphosulfate
  • PAP adenosine 3′,5′-diphosphate
  • the sulfation process can be coupled with an enzymatic regeneration of the 3′-phosphoadenosine-5′-phosphosulfate from the adenosine 3′,5′-diphosphate.
  • the enzymatic regeneration can employ an arylsulfotransferase as the catalyst and an aryl sulfate as a substrate.
  • preferred carbohydrate substrates can include GAGs, such as for example heparan sulfates, including heparin.
  • the arylsulfotransferase is a recombinant aryl sulfotransferase IV (AST-IV; e.g., rat AST-IV (SEQ ID NO:8)).
  • This enzyme when coupled to a sulfotransferase of choice, transfers sulfate from an aryl sulfate (e.g., p-nitrophenyl sulfate (PNPS) to PAP.
  • PNPS p-nitrophenyl sulfate
  • AST-IV exists in two oxidative forms (Marshall et al., (1997) J. Biol. Chem. 272, 9153-9160; Marshall et al., (1998) Chem .- Biol. Interact. 109, 107-116; Yang et al., (1998) Chem .- Biol. Interact. 109, 129-135; Yang et al. (1996) Protein Expression Purif. 8, 423-429; Guo et al. (1994) Chem .- Biol. Interact. 92, 25-31; Chen et al. (1992) Protein Expression Purif. 3, 421-6; Lin et al. (1998) Anal. Biochem.
  • AST IV can be cloned from a rat liver cDNA library, overexpressed in a recombinant bacterial system (e.g., E. coli ) and isolated (See, e.g., U.S. Pat. No. 6,255,088, herein incorporated by reference in its entirety).
  • a recombinant bacterial system e.g., E. coli
  • isolated See, e.g., U.S. Pat. No. 6,255,088, herein incorporated by reference in its entirety).
  • Coupling the sulfotransferase catalyzed sulfation reaction with a PAPS regeneration system can provide a further advantage of generating PAPS utilized in the reaction directly from PAP. That is, the reaction mixture can be formulated to combine PAP with a PAPS regenerating enzyme prior to or simultaneously with addition of a sulfotransferase to the reaction mixture. The PAPS regenerating enzyme can then generate PAPS from the PAP for use by the sulfotransferase, thereby alleviating the need of supplying any of the more expensive and unstable PAPS to the reaction mixture.
  • a method of sulfating a polysaccharide comprising providing a reaction mixture comprising therein adenosine 3′,5′-diphosphate (PAP), a PAPS regenerating enzyme and a sulfur donor compound (other than PAPS) and incubating the reaction mixture for a time period sufficient to catalyze the production of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) from the PAP by the PAPS regenerating enzyme utilizing the sulfur donor compound as a substrate.
  • PAP adenosine 3′,5′-diphosphate
  • PAPS a PAPS regenerating enzyme
  • sulfur donor compound other than PAPS
  • the method further comprises incubating a polysaccharide substrate and at least one O-sulfotransferase (OST) enzyme with the reaction mixture, wherein production of a sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to PAP and wherein the PAPS regenerating enzyme then catalyzes regeneration of the PAPS from the PAP, again utilizing the sulfur donor compound as a substrate.
  • OST O-sulfotransferase
  • kits for sulfating polysaccharides comprises at least one sulfotransferase enzyme (e.g., at least one OST); and a reagent which modifies adenosine 3′,5′-diphosphate (PAP) to reduce an inhibitory effect of PAP on the polysaccharide sulfation.
  • the at least one sulfotransferase enzyme is contained within a first container and the reagent is contained within a second container.
  • the kit can further comprise instructions for sulfating a polysaccharide.
  • the at least one sulfotransferase enzyme is an OST enzyme selected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST, and combinations thereof.
  • the OST enzyme is a recombinant OST enzyme, such as for example a recombinant OST enzyme produced in a bacterial expression system.
  • the OST enzyme is a fusion protein, such as for example a MBP-2-OST fusion protein or a MBP-6-OST fusion protein.
  • the OST enzyme is immobilized to a substrate, such as for example an agarose bead.
  • the reagent comprises a PAPS regeneration system comprising a PAPS regenerating enzyme (e.g., AST-IV) and a sulfur donor compound (e.g., PNPS).
  • a PAPS regenerating enzyme e.g., AST-IV
  • a sulfur donor compound e.g., PNPS
  • the reagent comprises a phosphatase enzyme (e.g., 3′-ribonucleotide phosphohydrolase).
  • CDSNS Heparin Chemically Desulfated N-Sulfated Heparin
  • heparin Treatment of heparin with a dimethyl sulfoxide/methanol mixture (9:1, v/v) at 80° C. for 24 h resulted in nearly complete N,O-desulfation with ⁇ 5% of the solvolytically resistant O-sulfo groups remaining.
  • Chemical N-sulfation with sulfur trioxide-pyridine afforded CDSNS heparin, which was characterized by disaccharide analysis and 1 H and two-dimensional COSY NMR.
  • 3-OST-1 bacterial expression plasmid (b3-OST-1-pET28).
  • the cDNA fragment encoding the catalytic domain of 3-OST-1 (G48-H311) was amplified from m3-OST-1-pcDNA3 with a 5′ overhang containing an NdeI site and a 3′ overhang containing an EcoRI site.
  • This construct was inserted into the pET28a vector (Novagen) using the NdeI and EcoRI restriction sites to produce a (His) 6 -tagged protein.
  • the resultant plasmid (b3-OST-1-pET28) was sequenced to confirm the reading frame and the lack of mutations within the coding region (University of North Carolina, DNA sequencing core facility).
  • the plasmid, b3-OST-1-pET28 was transformed into BL21(DE3)RIL cells (Stratagene, La Jolla Calif., U.S.A) for the expression of 3-OST-1.
  • NTA-agarose resin Qiagen, Valencia, Calif., U.S.A.
  • the resin was loaded onto a column and the protein was eluted with an imidizole gradient from 10 mM to 250 mM.
  • 3-OST-3 expression plasmid The cDNA fragment encoding the catalytic domain of 3-OST-3 (G139-G406) was amplified from plasmid h3-OST-3A-pcDNA3 (Liu, J. et al (1999) J. Biol. Chem. 274, 5185-5192) with a 5′ overhang containing an NdeI site and a 3′ overhang containing an EcoRI site. This construct was inserted into the pET28a vector (Novagen) using the NdeI and EcoRI restriction sites to produce a (His) 6 -tagged protein.
  • the resultant plasmid was sequenced to confirm the reading frame and the lack of mutations within the coding region (University of North Carolina, DNA sequencing core facility).
  • the plasmid was transformed into BL21 (DE3)RIL cells (Stratagene) for the expression of 3-OST-3.
  • NTA-agarose resin Qiagen
  • the resin was loaded onto a column and the protein was eluted with an imidizole gradient from 10 mM to 250 mM.
  • Immobilized Hs Sulfotransferases Preparation of Immobilized Hs Sulfotransferases. Dialyzed sulfotransferases (3 ml, 4 mg/ml) in phosphate-buffered saline buffer (3 mM KCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , 138 mM NaCl, pH 7.2) were mixed with 1 ml of AMINOLINK® plus beads (Pierce, Rockford, Ill., U.S.A.) following a protocol from the manufacturer. Immobilized enzyme was washed with 50 mM MES, 1% Triton X-100, 1 mM MgCl 2 , and 1 mM MnCl 2 , pH 7.0, and stored at 4° C.
  • phosphate-buffered saline buffer 3 mM KCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO
  • Immobilized proteins 100 ⁇ l, ⁇ 300 ⁇ g of immobilized enzyme
  • substrate 100 ⁇ g of substrate
  • [ 35 S]PAPS 1000 cpm/pmol
  • 1 ml of 50 mM MES, pH 7.0, 1% Triton X-100, 1 mM MgCl 2 , and 1 mM MnCl 2 100 ⁇ g of substrate
  • Triton X-100 1 mM MgCl 2
  • 1 mM MnCl 2 1 mM MnCl 2
  • the supernatant was collected, and the beads were washed with 3 ⁇ 200 ⁇ l of 1 M NaCl in 25 mM MOPS (pH 7.0).
  • the supernatant and washes were combined, diluted with 2 ml of water, and subjected to DEAE chromatography to determine the amount of [ 35 S]HS product used to determine the activities of the various HS O-STs.
  • N-terminal His 6 -tagged AST-IV was expressed in E. coli and purified as described by Burkat and colleagues (Burkart et al. (2000) J. Org. Chem. 65, 5565-5574) at a yield of ⁇ 50 mg/liter of bacterial culture.
  • the full-length cDNA of rat AST-IV was a generous gift of Dr. Michael Duffel (University of Iowa) (Sheng et al. (2004) Drug Metabol. Dispos. 32, 559-565).
  • Antithrombin (AT)-binding and gD-binding experiments utilized 35 S-labeled polysaccharides prepared using [ 35 S]PAPS.
  • 2 mg of HS substrate was incubated with 4 ml of beads with immobilized sulfotransferase ( ⁇ 12 mg of immobilized enzyme) at 25° C. in 20 ml of 50 mM MES, pH 7.0, 1% Triton X-100, 1 mM MgCl 2 , and 1 mM MnCl 2 , 200 ⁇ M [ 35 S]PAPS (1000 cpm/pmol) for 1 h.
  • the resultant polysaccharide was recovered using DEAE chromatography.
  • Disaccharide Analysis Synthesized polysaccharides (100 ⁇ g) were degraded by a mixture of heparin lyases as previously described (Moon et al. (2204) J. Biol. Chem. 279, 45185-45193) and desalted on BIOGEL® P-2 column (0.5 ⁇ 200 cm; Bio-Rad Labs) in 0.1 M NH 4 HCO 3 . Disaccharides were analyzed by a C 18 reversed phase column (0.45 ⁇ 25 cm; Vydac, Columbia, Md., U.S.A.) with UV 232 detection and identified by coelution with appropriate standards (Chen et al. (2003) Glycobiology 13, 785-794). The overall recovery yield of the disaccharide analysis was estimated by using 2-O-[ 35 S]heparin (100,000 cpm/70 ng; compound 2) as an internal control.
  • NMR Analysis Polysaccharide sample (1-2 mg) was dissolved in 0.5 ml of 2 H 2 O (99.9%), freeze dried to remove exchangeable protons, redissolved in 75 ⁇ l of 2 H 2 O (100.00%), and transferred to NMR microtubes (Shigemi, Inc., Allison Park, Pa., U.S.A.). NMR spectra were referenced relative to the HO 2 H at 4.80 ppm, and in COSY water was suppressed by presaturation of the HO 2 H resonance.
  • the equilibrium response unit RU(eq) values from the sensorgrams of FGF2 binding to polysaccharide-containing surfaces were used to construct Scatchard plots, RU(eq)/C versus RU(eq), where C is the free protein concentration, resulting in linear, first degree polynomial functions confirming the one-to-one binding of FGF-polysaccharide and to estimate binding affinity.
  • a two-state reaction model was applied to the AT-polysaccharide interactions measured by SPR using curve fitting to estimate the association and dissociation rate constants and affinity constant.
  • the BaF3 cells ectopically expressing FGFR1c have been previously described (Ornitz et al. (1996) J. Biol. Chem. 271, 15292-15297).
  • the BaF3-FGFR1c cells were maintained in RPMI 1640 medium (Sigma Chemical Co., St.
  • fetal bovine serum 0.5 ng/ml interleukin (IL)-3 (PeproTech Inc., Rocky Hill, N.J., U.S.A.), 2 mM L-glutamine, penicillin (50 IU/ml) and streptomycin (50 ⁇ g/ml), and 50 ⁇ M ⁇ -mercaptoethanol.
  • IL interleukin
  • BaF3 FGFR1c cells were washed three times with RPMI 1640 medium to remove IL-3 and resuspended in the growth medium lacking IL-3.
  • Concanavalin A-Sepharose (Sigma; 50 ⁇ l of 1:1 slurry) was then added, and the reaction was shaken at room temperature for 1 h. The beads were then washed by 3 ⁇ 1 ml of binding buffer, and the bound polysaccharide was eluted with 1 M NaCl.
  • AT was diluted with phosphate-buffered saline containing 1 mg/ml bovine serum albumin to give a stock solution at the concentration of 27 ⁇ M.
  • the chromogenic substrates, S-2765 (for factor Xa assay) and S-2238 (for thrombin assay) were from Diapharma (West Chester, Ohio, U.S.A.) and made up at 1 mM with 1 mg/ml POLYBRENE® (Sigma) in water.
  • the synthesized polysaccharide (compounds 5, 7, and 8; FIG.
  • the initial reaction rates as a function of concentration were used to calculate the IC 50 values.
  • concentrations of the synthesized polysaccharides were determined using Alcian blue as described by Bjornsson (Bjornsson (1993) Anal. Biochem. 210, 282-291) and quantitative disaccharide analysis as described above.
  • the Binding to Herpes Simplex Virus gD The assay for determining binding of 35 S-labeled polysaccharides (compounds 5 and 6; FIG. 1A ) to gD was carried out by an immunoprecipitation procedure using anti-gD monoclonal antibody (DL6) (Shukla et al. (1999) Cell 99, 13-22).
  • DL6 anti-gD monoclonal antibody
  • HS Sulfotransferases in E. coli .
  • the presently disclosed subject matter provides methods for synthesizing biologically active HS, such as for example FGF-binding HS (e.g., compound 4, including 4a and 4b), AT-binding HS (e.g., compound 5), and herpes simplex virus gD-binding HS (e.g., compound 6). See FIG. 1A .
  • FGF-binding HS e.g., compound 4, including 4a and 4b
  • AT-binding HS e.g., compound 5
  • herpes simplex virus gD-binding HS e.g., compound 6
  • FIG. 1A Four enzymes, including 2-OST, 6-OST, 3-OST-1, and 3-OST-3, were utilized for the syntheses of these particular targets.
  • Bacterial expressed 3-OST-1 and 3-OST-3 can exhibit substrate specificity and specific enzymatic activity comparable with those of their counterparts expressed in insect cells (Moon et
  • Immobilized Enzymes are Reusable. 2-OST, 6-OST, 3-OST-1, and 3-OST-3 were immobilized on agarose to be reusable and to enhance the thermal stability. Immobilized enzymes were incubated with polysaccharide substrate and PAPS for 1 h at room temperature. The sulfated polysaccharide product was separated from the immobilized enzyme by washing the beads with 1 M NaCl followed by centrifugation, making the immobilized enzymes ready for the next synthetic cycle. The cycle was repeated 10 times, after which each of the immobilized enzymes were assayed and showed >80% of their catalytic activity ( FIG. 2 ). It was also determined that the immobilized enzymes also maintained >65% of their catalytic activity after 2 months of storage at 8° C.
  • PAP inhibits sulfotransferase-catalyzed reactions.
  • a PAPS regeneration system can be used to convert PAP to PAPS by relying on AST-IV to catalyze the transfer of the sulfo group from PNPS to PAP, as illustrated in FIG. 1B .
  • the presently disclosed subject matter provides for the use of the PAPS regeneration system coupled with O-sulfotransferases. As disclosed herein, the PAPS regeneration system performed very well with 2-OST, 6-OST, and 3-OST-1. Complete modification of the substrate could be demonstrated by the low susceptibility of polysaccharide product to undergo additional sulfation using [ 35 S]PAPS with soluble enzymes, as disclosed herein above.
  • the modification by 2-OST elevated the level of ⁇ UA2S-GlcNS (disaccharide 4) by about 5-fold ( FIG. 3B and Table 2), confirming the structure of compound 2.
  • Analysis of compound 3 afforded a 7-fold increase in the level of the disaccharide ⁇ UA-GlcNS6S (disaccharide 3) compared with compound 1, consistent with 6-OST-catalyzed modification ( FIG. 3C and Table 2).
  • the level of trisulfodisaccharide, ⁇ UA2S-GlcNS6S (disaccharide 5), in compounds 4a and 4b was increased by about 10-fold compared with that of compound 1 ( FIGS. 3D and 3E , and Table 2).
  • Each synthesized polysaccharide (100 ⁇ g) was digested with a mixture of heparin lyases.
  • the resultant disaccharides were purified by a BioGel P-2 column and resolved by RPIP-HPLC.
  • the integral of GlcNS-H6a and IdoUA2S-H2 was compared with the integral of the N-acetyl methyl group in compound 4a to estimate the level of incorporation of the 6-O-sulfo group. Integration showed that the 6-O-sulfo group was incorporated into 25% of the GlcNS residues. The reduced level of incorporation is not surprising, since only the 6-OST-1, and not 6-OST-2 and -3, was used in this synthesis.
  • the incorporation of the 3-O-sulfo group was calculated by comparing the integral of the N-acetyl methyl group with GlcN-H3 and IdoUA-H4 (overlapping signal).
  • composition estimated by NMR is consistent with the results of disaccharide analysis.
  • NMR analysis suggests that about 15% of the glucosamine unit is N-acetylated, which is similar to the results of disaccharide analysis for ⁇ UA-GlcNAc (14-22%; Table 2).
  • NMR analysis demonstrates that compound 2 comprises 35% of IdoUA2S-GlcNS, and compound 4a comprises 25% of IdoUA2S-GlcNS6S, and disaccharide analysis demonstrates that compounds 2 and 4a comprise 40% of ⁇ UA2S-GlcNS and 30% of ⁇ UA2S-GlcNS6S, respectively. Because of the signal overlap, the degree of 6-O-sulfation in compounds 3 and 4b using 1 H NMR was not calculated.
  • the BaF3 FGFR1c cells normally depend on IL-3 for growth. In the absence of IL-3, the cell proliferation depends on the addition of both FGF and heparin or HS (Ornitz et al. (1996) J. Biol. Chem. 271, 15292-15297). The activity of compounds 1, 2, 3, 4a, and 4b and heparin in promoting cell mitogenesis was measured using the FGF-2/FGFR1c system in BaF3 cells as described herein above.
  • the cells receiving compounds 4a and 4b showed an increase in [ 3 H]thymidine incorporation, which was about 60 and 40% of that of heparin, respectively, suggesting that the combinations of 2-O- and 6-O-sulfations confer the activity in promoting cell proliferation ( FIG. 4A ).
  • the activity of 4a and 4b was also compared with that of heparin at different concentrations ( FIG. 4B ). It was found that compound 3 had moderate activity in promoting cell proliferation, whereas compounds 1 and 2 did not exhibit activity. The results are consistent with previously reported data on the contributions of the sulfo groups of HS to the mitogenic activity (Guimond and Turnbull (1999) Curr. Biol.
  • the Anticoagulant Activity of the Synthesized Polysaccharides Heparin achieves its anticoagulant activity by forming a 1:1 complex with AT, which inhibits the activities of factor Xa and thrombin (Rosenberg et al. (1997) J. Clin. Invest. 99, 2062-2070). Because it is known that the introduction of the 3-O-sulfo group by 3-OST-1 is essential for the synthesis of anticoagulant HS, different types of 3-O-sulfo group-containing polysaccharides, compounds 5, 7, and 8, were prepared and their activities in inhibiting factor Xa and thrombin tested (Table 4).
  • heparin is a potent activator for AT-mediated inhibition of factor Xa and thrombin
  • ARIXTRA® GaxoSmithKline
  • Compound 5 has very similar potency to heparin, inhibiting the activities of both factor Xa and thrombin, suggesting that the presently disclosed enzyme-based approach is indeed capable of synthesizing the anticoagulant polysaccharide. It has been reported that the presence of 2-O-sulfo groups is not essential for HS binding to AT and its resulting anticoagulant activity (Zhang et al.
  • polysaccharide intermediate compound 8 lacks 2-O-sulfo groups but still exhibits anticoagulant activity, consistent with this previous report (Zhang et al. (2001) J. Biol. Chem. 276, 28806-28813).
  • compound 7 lacks 6-O-sulfo groups and, thus, has no anticoagulant activity, since 6-O-sulfo groups are critical in AT binding (Atha et al. (1985) Biochemistry 24, 6723-6729).
  • Another 3-O-sulfated polysaccharide (compound 6) was also prepared to test for its anti-Xa and antithrombin activities.
  • both compounds 5 and 6 carry a 3-O-sulfoglucosamine unit, although it is located in different saccharide sequences ( FIG. 1A ). It is known that 3-OST-3-modified HS does not bind to AT (Liu et al. (1999) J. Biol. Chem. 274, 5185-5192). As expected, compound 6 does not exhibit any anti-Xa and antithrombin activities. The binding of AT to the synthesized compounds was also measured (Table 4). It is clear that the anticoagulant activities of the compounds correlated to their binding affinity to AT. Taken together, these results demonstrate that the anticoagulant activities of these enzymatically synthesized polysaccharides are consistent with the known structure activity relationship of HS.
  • c Heparin was from Sigma.
  • d ND not determined.
  • e Heparan sulfate was isolated from bovine kidney.
  • f Arixtra is the chemically synthesized antithrombin-binding pentasaccharide, which was obtained from a local pharmacy.
  • Herpes simplex virus utilizes HS as a receptor to infect the target cells (Shukla and Spear (2001) J. Clin. Invest, 108, 503-510).
  • a specific 3-O-sulfo group-containing HS generated by 3-OST-3, -5, or -6, serves as an entry receptor for herpes simplex virus type 1 through HS binding of gD (Shukla et al. (1999) Cell 99, 13-22; Xia et al. (2002) J. Biol. Chem. 277, 37912-37919; Xu et al. (2005) Biochem. J. 385, 451-459).
  • HS including heparin
  • the presently disclosed subject matter provides an approach to synthesize sulfo group-containing HS polysaccharides that have desired biological activities, including for example HS that binds to FGF2, herpes simplex virus gD, or AT.
  • These HS polysaccharides can also demonstrate appropriate biological activity, such as anticoagulant activity mediated through AT binding and the activity in promoting cell proliferation.
  • the presently disclosed subject matter permits the synthesis of greater than 1 mg amounts of specific sulfo group-containing polysaccharides, sufficient for testing their activities in biochemical and biological assays.
  • the quantities synthesized are also sufficient for extensive structural analysis, including disaccharide analysis and one- and two-dimensional NMR analysis.
  • examples 1-3 two approaches were used to increase the scale of enzymatic synthesis.
  • HS sulfotransferases were expressed in E. coli , readily affording 20-50 mg of purified enzymes. It has been shown that both 3-OST-1 and 3-OST-3 can be expressed in E. coli in relatively high yield.
  • the presently disclosed subject matter also provides for the expression in E. coli of both 2-OST and 6-OST as the soluble 2-OST and 6-OST-MBP fusion proteins.
  • efficiency of enzymatic sulfation was improved by utilizing immobilized O-sulfotransferases and coupling their use to a PAPS regeneration system.
  • PAP inhibits HS O-sulfotransferase activities with IC 50 values of ⁇ 100 ⁇ M under the reaction conditions used in the presently disclosed syntheses, making milligram-scale synthesis difficult without continuously removing PAP.
  • a PAPS regeneration system would permit use of PNPS as the sulfo donor and require only catalytic amounts of PAP, significantly reducing the cost of synthesis.
  • the listed price of PNPS in the Aldrich catalog is about 300-fold less than PAPS.
  • the immobilized enzyme format facilitates use of the enzymes repeatedly, making the method amendable to a large scale synthesis, since scale-up of immobilized enzyme columns is generally known in the art.
  • HS is believed to be present in block structures, comprising highly sulfated and non-sulfated domains (Gallagher, J. T. (2001) J. Clin. Invest 108, 357-361), and the HS with the biological activities largely contain the highly sulfated domains. Without wishing to be limited by theory, it is believed that the modifications in the present system indeed happen in a block fashion based on the following facts. First, the results of the disaccharide analysis concluded that both 6-O-sulfation and 2-O-sulfation are carried out predominantly in the sulfated region ( FIG. 3 and Table 2).
  • the enzymatically-modified products exhibit the anticipated biological functions, including the activation of FGF/FGF receptor signaling (compounds 4a and 4b), carrying anticoagulant activity (compound 5), and binding to herpes simplex virus glycoprotein D (compound 6). Because the HS carrying these functions must contain the domain structures with a size larger than pentasaccharide (Petitou and van Boeckel (2004) Angew. Chem. Int. Ed. 43, 3118-3133; Atha et al. (1985) Biochemistry 24, 6723-6729; Liu et al. (2002) J. Biol. Chem. 277, 33456-33467; Maccarana et al. (1993) J. Biol. Chem. 268, 23898-23905), the synthetic products with the desired functions suggest that the modifications indeed occur in a block fashion.
  • the presently disclosed subject matter provides a method for enzymatic sulfation and preparation of HS with distinct biological activities.
  • Unique sulfated saccharide sequences play a dominant role in the function and specificity of HS/heparin.
  • the presently disclosed methods demonstrate the capability of using a collection of HS biosynthetic enzymes to synthesize HS/heparin with selected biological activities.
  • the synthetic scale with this method can be easily increased for large scale synthesis, provided that both the enzymes and the sulfo donor are easily accessible.
  • the current method clearly demonstrates that HS/heparin having specific biological activities can be synthesized by subjecting a backbone saccharide polymer to different enzymatic modifications.

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US20110217249A1 (en) * 2010-03-03 2011-09-08 Frank Dreher Compositions and Methods for the Treatment of Skin Diseases and Disorders Using Antimicrobial Peptide Sequestering Compounds
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