WO2003029261A9 - Polymeres de glycosaminoglycane et procedes de preparation et d'utilisation de ces derniers - Google Patents

Polymeres de glycosaminoglycane et procedes de preparation et d'utilisation de ces derniers

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Publication number
WO2003029261A9
WO2003029261A9 PCT/US2002/022386 US0222386W WO03029261A9 WO 2003029261 A9 WO2003029261 A9 WO 2003029261A9 US 0222386 W US0222386 W US 0222386W WO 03029261 A9 WO03029261 A9 WO 03029261A9
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WIPO (PCT)
Prior art keywords
functional acceptor
providing
polymer
sugars
synthase
Prior art date
Application number
PCT/US2002/022386
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English (en)
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WO2003029261A2 (fr
WO2003029261A3 (fr
Inventor
Paul L Deangelis
Original Assignee
Paul L Deangelis
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/142,143 external-priority patent/US7307159B2/en
Application filed by Paul L Deangelis filed Critical Paul L Deangelis
Priority to AU2002361549A priority Critical patent/AU2002361549A1/en
Priority to EP02797026A priority patent/EP1470235A4/fr
Publication of WO2003029261A2 publication Critical patent/WO2003029261A2/fr
Publication of WO2003029261A9 publication Critical patent/WO2003029261A9/fr
Publication of WO2003029261A3 publication Critical patent/WO2003029261A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to methodology for the production of polymers, such as oligosaccharides, by a glycosaminoglycan synthase and, more particularly, polymer production utilizing glycosaminoglycan synthases from Pasteurella multocida.
  • oligosaccharides small sugar chains
  • Complex sugars cause biological effects by binding to target proteins including enzymes and receptors.
  • the presently claimed and disclosed invention also relates to a chemoenzymatic synthesis methodology to create both pure, chimeric, and hybrid polymers, such as oligosaccharides, composed of hyaluronan, chondroitin, keratan, dermatan, heparin units, and combinations
  • oligosaccharides are used in parallel using a microarray format or a microtiter
  • Target proteins implicated in diseases are thereafter tested for their ability to bind to microarrays or microtiter plates and thus have
  • Polysaccharides are large carbohydrate molecules comprising from about
  • the polysaccharide's biological function is due to the interaction of the
  • polysaccharide with proteins such as receptors and growth factors.
  • glycosaminoglycan class of polysaccharides and oligosaccharides which includes heparin, chondroitin, dermatan, keratan, and hyaluronic acid, plays major roles in determining cellular behavior (e.g. migration, adhesion) as well
  • oligosaccharides are, therefore, essential for the correct formation and maintenance of the organs of the human body.
  • Pasteurella multocida produce authentic hyaluronic acid capsules, and other Pasteurella multocida (Type F and D) and pathogenic Escherichla coli (K4 and
  • the pathogenic microbes form the polysaccharide
  • Enzymes alternatively called synthases, synthetases, or transferases, catalyze the polymerization of polysaccharides found in living organisms.
  • cDNA complementary DNA
  • heteropolysaccharides utilize one UDP-sugar binding site to transfer both precursors, or alternatively, if there exists two dedicated regions for each
  • Vertebrate tissues and pathogenic bacteria are the sources of more exotic polysaccharides utilized in the medical field - e.g. as
  • hyaluronic acid from rooster combs are employed in several applications including clot prevention and eye surgery, respectively.
  • bacterial capsules e.g. various Streptococcus pneumoniae strains
  • Streptococcus pneumoniae strains are utilized to vaccinate both children and adults against disease with
  • polysaccharides are only available having a large molecular weight distribution
  • polymers in vitro utilize: (i) difficult, multistep sugar chemistry, or (ii) reactions
  • polysaccharides are the most abundant biomaterials on
  • Hyaluronic acid or "HA” is a linear polysaccharide of the
  • glycosaminoglycan class and is composed of up to thousands of ⁇ (l,4)GlcUA-
  • HA is a major structural element of the
  • HA has a high
  • HA also interacts with proteins
  • HA has been widely used as a viscoelastic replacement for the vitreous humor of the
  • HA injection directly into joints is also used to alleviate pain
  • HA is also made by certain microbes that cause disease in humans and
  • Some bacterial pathogens namely Gram-negative Pasteurella
  • multocida Type A and Gram-positive Streptococcus Group A and C produce an extracellular HA capsule which protects the microbes from host defenses such
  • Mutant bacteria that do not produce HA capsules are 10 2 - and
  • PBCV-1 Paramecium bursaria Chlorella virus
  • HAS HA synthases
  • HASs are membrane proteins localized to the lipid bilayer at the cell surface.
  • the HA polymer is transported across the bilayer into the extracellular space.
  • HASs a single species of polypeptide catalyzes the
  • HasA (or spHAS) from Group A Streptococcus pyogenes was the first HA
  • HAS3 and the viral enzyme, A98R are quite similar at the amino acid level to certain regions of the HasA polypeptide chain ( ⁇ 30% identity overall) and were
  • cytoplasmic central domain ( ⁇ 200 amino acids).
  • the amino terminal region appears to contain two transmembrane segments, while the carboxyl terminal region appears to contain three to five membrane-associated or
  • transmembrane segments depending on the species. Very little of these HAS polypeptide chains are expected to be exposed to the outside of the cell.
  • streptococcal enzyme was reported to add sugars to the nonreducing terminus
  • chondroitin synthase also is useful, but it adds chondroitin chains to the acceptor's non-reducing
  • the present invention provides a method for the production of polysaccharides or oligosaccharides of HA, chondroitin, and chimeric or hybrid molecules incorporating both HA and chondroitin.
  • the present invention also encompasses the use of modified synthases
  • the present invention also encompasses the methodology of
  • polysaccharide or oligosaccharide polymer grafting i.e. HA or chondroitin
  • a hyaluronan synthase (pmHAS) or a chondroitin synthase (pmCS) or a heparin synthase (pmHS, PglA) from various types of P. multocida.
  • pmHAS hyaluronan synthase
  • pmCS chondroitin synthase
  • pmHS, PglA heparin synthase
  • graft on polysaccharides of various size and composition can also be utilized to graft on polysaccharides of various size and composition.
  • Such grafting methodologies have uses, but are not limited thereto, for the production of sugar libraries (both natural and chimeric or hybrid), protein-testing or cell-testing in microarray or microtiter plate formats.
  • pmHAS A unique HA synthase, from the fowl cholera pathogen, Type A P.
  • multocida has been identified and cloned and is disclosed and claimed in co-
  • the pmHAS is an authentic HA synthase.
  • PglA and pmHS Two unique heparin synthases, PglA and pmHS, from Type A, D, and F P. multocida and Type D P. multocida, respectively, have been identified and cloned and are disclosed and claimed in co-pending U.S. Serial No.10/142, 143, filed May 8, 2002, and entitled "Heparin/Heparosan Synthase from P. multocida and Methods of Making and Using Same", the contents of which are hereby expressly incorporated herein in their entirety.
  • proteins allows E. coli host cells to produce enzymes that polymerize heparosan chains.
  • Laboratory strains of E. coli normally do not make heparin. Extracts of recombinant E. coli, when supplied with the appropriate UDP-sugars, make heparin in vitro.
  • the PglA and the pmHS are authentic heparin synthases.
  • pmHAS recombinant pmHAS, pmHS, PglA, and pmCS synthases add sugars to the nonreducing end of a growing polymer chain.
  • the correct monosaccharides are added sequentially in a stepwise fashion to the nascent chain or a suitable exogenous , HA or chondroitin oligosaccharide acceptor molecule.
  • the pmHAS sequence is significantly different from the other known HA synthases. There appear to be only two short potential sequence motifs ([D/N]DGS[S/T] (SEQ ID N072); DSD[D/TJY (SEQ ID NO:73)) in common between pmHAS and the Group A HAS-spHAS. Instead, a portion of the central region of the pmHAS is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular
  • pmHAS is about twice as long as any other HAS enzyme.
  • pmHS or PglA are supplied with functional acceptor oligosaccharides, total HA,
  • chondroitin and heparin biosynthesis is increased up to 50-fold over reactions without the exogenous oligosaccharide.
  • the native versions of the pmHAS, pmCS, pmHS, and PglA enzymes isolated from P. multocida do not perform
  • the nature of the polymer retention mechanism of the pmHAS, pmCS, pmHS, and PglA polypeptide might be the causative factor for
  • a HA- or chondroitin- or heparin-binding site may exist that holds onto the HA or chondroitin or heparin chain during polymerization.
  • HA or chondroitin or heparin oligosaccharides supplied by the hand of man are also capable of occupying this site of the recombinant enzyme and thereafter be
  • HAS enzyme from Group A and C Streptococcus bacteria has been detergent-solubilized and purified in an active state in small quantities.
  • streptococcal enzyme Once isolated in a relatively pure state, the streptococcal enzyme has very limited stability. A soluble recombinant form of the HAS enzyme from P.
  • pmHAS 1"703 comprises residues 1-703 of the 972 residues of the native pmHAS enzyme.
  • pmHAS 1"703 can be mass-produced in E. coli and
  • the pmHAS 1"703 enzyme retains the ability of the
  • parent enzyme to add onto either a long HA polymer, a short HA primer, a long
  • chondroitin polymer a short chondroitin primer, a short chondroitin polymer, as
  • the purified pmHAS 1"703 enzyme is stable in an optimized buffer for days on ice and for hours at normal reaction temperatures.
  • One formulation of the optimal buffer consists of 1M ethylene glycol, 0.1 - 0.2 M ammonium sulfate, 50mM Tris, pH 7.2, and protease inhibitors which also allow the stability and specificity at typical reaction conditions for sugar transfer.
  • UDP-sugars and divalent manganese (10-20 mM) are added for the reaction.
  • pmHAS 1"703 will also add a HA polymer onto plastic beads with an immobilized short HA primer or any other substrate capable of having an acceptor molecule or acceptor group thereon.
  • pmCS, pmHAS, pnHS, and PglA possess two separate glycosyltransferase sites. Protein truncation studies demonstrated that residues 1-117 of pmHAS can be deleted without affecting catalytic activity; similar truncation of the homologous pmCS, pmHS, and PglA enzymes may also be preferred.
  • the carboxyl-terminal boundary of the GlcUA-transferase of pmHAS resides within residues 686-703 and within residues 686-704. of pmCS.
  • the highly homologous (90% identical) pmCS can also be mutated in the same
  • pmCS results in an enzyme with only GalNAc-transferase activity.
  • Type F P. multocida synthesizes an unsulfated chondroitin ( ⁇ 3N- acetylgalactosamine [GalNAc]- ⁇ 4GlcUA) capsule. Domain swapping between pmHAS and the homologous chondroitin synthase, pmCS, has been performed.
  • a chimeric or hybrid enzyme consisting of residues 1-427 of pmHAS and
  • polypeptide as well as further delineate the site boundaries of both enzymes.
  • the hexosamine-transferase site resides in the N-terminal domain while the
  • GlcUA-transferase site' resides in the COOH-terminal domain of these GAG
  • the present invention encompasses methods of producing a variety of
  • the present invention also incorporates the propensity of certain recombinant enzymes, when prepared in a virgin state, to utilize various acceptor molecules as the seed for further polymer growth: naturally occurring forms of the
  • the present invention results in (a) the production of hybrid oligosaccharides or polysaccharides and (b) the formation of polysaccharide
  • Such hybrid polymers can serve as "molecular glue” — i.e. when two
  • Such polysaccharide coatings are useful for integrating a foreign object within a surrounding tissue matrix.
  • a prosthetic device is more firmly attached to the body when the device is coated with a naturally adhesive
  • the device's artificial components could be masked by the biocompatible coating to reduce immunoreactivity or inflammation.
  • Another aspect of the present invention is the coating or grafting
  • GAGs onto various drug delivery matrices or bioadhesives or suitable
  • medicaments to improve and/or alter delivery, half-life, persistence, targeting
  • pmHAS and pmCS and pmHS and PglA to elongate defined oligosaccharide derivatives.
  • the non-reducing end sugar addition allows the reducing end to be modified for other purposes; the addition of GAG chains to small molecules,
  • pmHAS and pmCS and pmHS and PglA appear distinct from most other known HA and chondroitin and heparin synthases based on differences in sequence, topology in the
  • pmHAS 1"703 is a
  • pmHAS 1"650 is inactive as a HA
  • mutants substituted at residue 196 possessed GlcUA- transferase activity while those substituted at residue 477 possessed GlcNAc-
  • Pasteurella multocida Type F the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor.
  • Serator No. 09/842,484 filed April 25, 2002, and entitled "Chondroitin Synthase Gene and Methods of Making and Using Same"
  • chondroitin AC lyase It was found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. A Type F polysaccharide synthase was molecularly cloned and its enzymatic activity was characterized. The 965-
  • pmCS residue enzyme
  • glycosyltransferase that forms a polysaccharide composed of chondroitin
  • multocida also are a single polypeptide specie that possess both transferase activities to catalyze heparin/heparosan.
  • NanoHA N-(nanoHA).
  • nanoHA signifies the very small
  • HA oligosaccharides are also very promising in an in vivo
  • HA oligosaccharides susceptible to the therapeutic action of HA oligosaccharides.
  • a very desirable attribute of HA-oligosaccharides for therapeutics is that
  • metastasis are HA chains composed of 10 to 14 sugars.
  • oligosaccharides are extremely limited and will not allow the medical potential
  • the enzyme wants to degrade the polymer to the 4 sugar end stage product, but this sugar is inactive for cancer treatment.
  • the use of acid hydrolysis also removes a fraction of the acetyl groups from the
  • the partial depolymerization method only yields fragments of the original
  • HA polymer and is essentially useless for creating novel sugars beyond simple derivatizations (e.g. esterifying some fraction of GlcUA residues in an
  • hyaluronic acid or chondroitin or heparin enzyme catalysts and substrates are examples of hyaluronic acid or chondroitin or heparin enzyme catalysts and substrates.
  • FIG. 1 is a graphical representation showing that an HA tetramer stimulates pmHAS polymerization.
  • FIG. 2 is a graphical plot showing that HA polymerization is effected by HA oligosaccharides.
  • FIG. 3 is a graphical plot showing HA tetramer elongation into larger
  • FIG. 4 is a graphical representation of a thin layer chromatography
  • FIG. 5 is a graphical representation of thin layer chromatography analysis
  • FIG. 6 is an electrophoresis gel showing the purification of pmHAS 1"703 .
  • FIG. 7 is a pictorial representation of the pmHAS truncation mutants.
  • FIG. 8 is a graphical representation of a mutant combination assay.
  • FIG. 9 is a tabular representation showing enzyme activity of the pmHAS-
  • FIG. 10 is a graphical representation of a high-throughput assay for
  • FIG. 11 is a Western Blot analysis showing the expression of pmHAS and its truncated forms. Either whole cell lysates (pmHAS 437"972 , pmHAS 1"567 , and pmHAS 152"756 ) or membrane preparations (pmHAS 437"756 , pmHAS 1"567 , rl-972, nl- 972) or B-Per extract (pmHAS 1"703 ) were analyzed by Western blot (r,recombinant from E. coli; n, native from P-1059). The bars on the left denote the position of molecular weight standards (from top to bottom: 112, 95, 55, and 29 kDa).
  • FIG. 12 is a pictorial representation of domains Al and A2 of pmHAS.
  • A The approximate relative positions of domain Al and A2 in pmHAS and pmHAS 1'703 .
  • B Partial alignment of the amino acid sequences of the two domains (residue 161-267 and 443-547).
  • Al in Figure 12 (B) corresponds to residues 161-267 of SEQ ID NO: 2.
  • A2 in Figure 12 (B) corresponds to residues 443-547 of SEQ ID NO: 2.
  • the aspartate residues mutated in our studies were marked with *. Identical residues are in bold.
  • FIG. 13 is a graphical representation of the complementation of the HAS activity of mutant enzymes in vitro.
  • HAS enzyme assays with HA-derived acceptor were performed in the presence of either wild type pmHAS 1"703 alone, or D196 mutant alone, or D477 mutant alone or in the presence of both D196 and D477 mutants, for either 25 minutes (open bars) or 1.5 hours (solid bars).
  • FIG. 14 is a pictorial representation of a model of the two putative glycosyltransferase sites and the potential membrane association region of the pmHAS polypeptide.
  • FIG. 15 is a sequence alignment of pmCS and pmHAS.
  • the pmCS sequence is SEQ ID NO:4; and the pmHAS sequence is SEQ ID NO:9.
  • the two Pasteurella GAG synthases are highly homologous. Identical residues are denoted with the hyphen. The numbering scheme corresponds to the slightly
  • domains correspond to regions important for hexosamine transferase
  • FIG. 16 is a Western Blot Analysis of Truncated Recombinant Pasteurella GAG Synthases. Immunoreactive bands at the predicted size of 80 kDa
  • V vector control
  • Std Prestained standards
  • FIG. 17 is a graphical plot of gel filtration analysis of radiolabeled polymer
  • the pmCS 1"704 extract (1 mg total protein) was incubated with chondroitin acceptor oligosaccharide (5 mg), UDP-[ 14 C]GlcUA and UDP- [ 3 H]GalNAc (580 mM, 0.16 mCi each) in a reaction volume of 200 ml for 30
  • dotted line measured by the in-line detector is presented as disintegrations per second (dps).
  • the double-headed arrow corresponds to a response of 20 dps.
  • A untreated polymer, peak 15.9 min;
  • B Flavobacterium chondroitinase AC lyase-treated polymer, peak, 19.2 min;
  • C HA lyase-treated polymer, peak 15.9 min.
  • the polymer peak with a size of ⁇ 100 to 400 kDa contained both radiolabeled sugars at a 1:1 ratio and was degraded only by the appropriate enzyme, chondroitin AC lyase.
  • FIG. 18 is a pictorial representation of a model of the two putative glycosyltransferase sites of pmHAS and pmCS.
  • PmHAS and pmCS contain two distinct and relatively independent glycosyltransferase sites. Each site possesses a DGS and a DXD amino acid motif.
  • a WGGED motif (SEQ ID NO: 74) is found near the junction of the two domains, and is involved in hexosamine-transferase activity.
  • the carboxyl-terminus is involved in membrane association (MEM ASSOC), but is not required for catalytic activity.
  • Residues 1-117 appear dispensable for catalysis of sugar transfer but may contain structure scaffolding or play other roles.
  • FIG. 19 graphically depicts Sequence Similarity of pmHS with KfiA and KfiC.
  • Elements of the Pasteurella heparosan synthase, HS1 (containing residues 91-240) and HS2 (containing residues 441-540) are very similar to portions of two proteins from the E. coli K5 capsular locus (A, residues 75- 172 of KfiA; C, residues 262- 410 of KfiC) as shown by this modified Multalin
  • the HS1 and HS2 elements may be important for hexosamine transferase or for glucuronic acid transferase activities, respectively, (con, consensus symbols: asterisks, [K or R] and [S or TJ; %, any one of F,Y,W; $, any one of L,M; !, any one of IN; #, any one of E,D,Q, ⁇ ).
  • the HS1 sequence is SEQ ID NO:64; the KfiC sequence is SEQ ID NO:80; the HS2 sequence is SEQ ID NO:63; and the KfiA sequence is SEQ ID NO: 81.
  • FIG. 20 depicts pmHS Activity Dependence on Acceptor and Enzyme Concentration.
  • Various amounts " of crude membranes containing the full-length enzyme, pmHSl-617 were incubated in.50 ⁇ l of buffer containing 50 mM Tris, pH 7.2, 10 mM MgCI 2 , 10 mM MnCI 2 , 500 ⁇ M UDP-[ 14 C]GlcUA (0.15 ⁇ Ci), and 500 ⁇ M UDP-GlcNAc.
  • Three parallel sets of reactions were performed with either no acceptor ( circles) or two concentrations of heparosan polymer acceptor (uronic acid: 0.6 ⁇ g, squares; 1.7 ⁇ g, triangles).
  • FIG. 21 Gel Filtration Analysis of Radiolabeled Polymer Synthesized in vitro.
  • the crude membranes containing pmHS (0.7 mg total protein) were incubated with UDP-[ 14 C]GlcUA and UDP- [ 3 H]GlcNAc (each 500 ⁇ M, 0.4 ⁇ Ci) in a 200 ⁇ l reaction volume either in the presence ( top panel) or absence (bottom
  • acceptor polymer (1 ⁇ g uronic acid). After various reaction times (denoted on curves: 20, 60, or 270 min), portions of the samples (75%) were analyzed on the PolySep column (calibration elution times in. minutes: void volume, 9.8; 580 kDa dextran, 12.3; 145 kDa dextran, 12.75, totally included volume, 16.7). The starting acceptor polymer eluted at 12.8 min. Large polymers composed of both radiolabeled sugars ( 14 C, C; 3 H, H) in an equimolar ratio were synthesized by pmHS.
  • FIG. 22(A-D) graphically depicts the alignment of the pmHS (two clones: A2, B10) with PglA, KfiA, KfiC, and DcbF.
  • the KfiC sequence is SEQ ID NO:80;
  • the HSA1 sequence is SEQ ID NO:83;
  • the KfiA sequence is SEQ ID NO:84;
  • the HSA2 sequence is SEQ ID NO:85.
  • the pmHS sequence is SEQ ID NO:70; the pglA sequence is SEQ ID NO:8; and the DcbF sequence is SEQ ID NO:61.
  • the A2 sequence is SEQ ID NO:6; the B10 sequence is SEQ ID NO:70; the pglA sequence is SEQ ID NO:8; and the DcbF sequence is SEQ ID NO:61.
  • the pmHS sequence is SEQ ID NO:70; the pglA sequence is SEQ ID NO:8; and the DcbF sequence is SEQ ID O:61.
  • FIG. 23 depicts chimeric constructs of pm-EG, pm-FH, pm-IK, and pm-JL. PCR-overlap-extension was performed.
  • Pm-EG contains residues 1-265 from pmHAS and residues 259-704 from pmCS and is a GlcUA-Tase.
  • Pm-FH contains residues 1-258 from pmCS and residues 266-703 from pmHAS and is an active chondrotin synthase.
  • Pm-IK contains residues 1-221 from pmHAS and residues 215-704 from pmCS and is a Glc-UA-Tase.
  • Pm-JL contains residues 1-214 from pmCS and residues 222-703 from pmHAS and is an active HA synthase.
  • the switch of Gal-NAc-transferring activity into GlcNAc-transferring activity indicated that 222-265 of pmHAS and possibly the corresponding residues 215-258 of pmCS play critical role in the selectivity between binding and/or transferring of GalNAc and GlcNAc substrate.
  • FIG. 24 depicts a comparison of partial primary sequences of pmHAS and different pmCSs.
  • Primary sequences of presumably chondroitin synthases from different Type F Pasteruella multocida were obtained by directly sequencing the products of colony-lysis PCR.
  • the MULTALIN alignment indicates that most of the differences between pmHAS and pmCS are conserved among these independent strains.
  • Residues that were substituted in site-mutagenesis studies were underlined.
  • the mutant polypeptides contain a single or combination of different mutations, indicated by star(s). None of these mutations changes the specificity of the original enzymes.
  • the PmHAS sequence is SEQ ID NO:86; the PmCS sequence is SEQ ID NO:87; the Turkey sequence is SEQ ID NO:88; the goose sequence is SEQ ID NO:89; and the Sea-lion sequence is SEQ ID NO:90.
  • FIG. 25 depicts chimeric constructs of pmHAS ⁇ -CS ⁇ ⁇ -HAS 266"703 and pmCS 1"214 -HAS 222"265 -CS 258"704 .
  • Pm-FH and pPm7A DNA were used to create pmHAS 1"221 -CS 215 - 258 -HAS 266"703 .
  • a very interesting result was that pmCS 1"214 - HAS 222"265 -CS 258"704 can transfer both GalNAc and GlcNAc to HA oligomer acceptor; this enzyme displays relaxed sugar specificity.
  • FIG. 26 depicts a summary of enzyme activities of chimeric proteins.
  • the enzymes are drawn as bars. Black bars represent pmCS.
  • White bars represent pmHAS. +, active; -, inactive.
  • PmCHC represents pmCS 1"214
  • FIG. 27 depicts a mass spectra of F-HA12 product. This fluorescent HA oligosaccharide was synthesized using the twin bioreactor scheme. A peak with
  • FIG. 28 is a pictorial representation of a hyalose biocatalytic scheme depicting the step-wise addition of sugars.
  • GAGs Glycosaminoglycans
  • Hyaluronan [HA], chondroitin, and heparan sulfate/heparin contain a uronic acid as the other component of the
  • the GAGs are
  • Vertebrates may contain all four types of GAGs, but the polysaccharide chain is often further modified after sugar polymerization.
  • One or more of GAGs may contain all four types of GAGs, but the polysaccharide chain is often further modified after sugar polymerization.
  • chondroitin and heparan sulfate/heparin chains in vertebrates are initially synthesized by elongation of a xylose-containing linkage tetrasaccharide attached to a variety of proteins. Keratan is either O-linked or N-linked to certain proteins depending on the
  • HA and all of the known bacterial GAGs are not part of the classification of proteins known as glycoproteins. All GAGs except HA are found
  • proteoglycan covalently linked to a core protein, and such combination is referred to as a proteoglycan.
  • Glycoproteins are usually much smaller than proteoglycans and
  • proteoglycan is also usually a glycoprotein, therefore usually contains other
  • GAGs and their derivatives are currently used in the medical field as ophthalmic and viscoelastic supplements, adhesion surgical aids to prevent post-operative adhesions, catheter and device coatings, and anticoagulants.
  • tissue engineering matrices immune and neural cell modulators, and drug
  • GAGs Complex carbohydrates, such as GAGs, are information rich molecules.
  • a major purpose of the sugars that make up GAGs is to allow communication
  • proteins bind to particular sugar chains in a very selective fashion.
  • a protein may simply adhere to the sugar, but quite often the protein's
  • intrinsic activity may be altered and/or the protein transmits a signal to the cell to modulate its behavior.
  • the protein transmits a signal to the cell to modulate its behavior.
  • heparin binding to inhibitory proteins helps shuts down the clotting response.
  • HA binds to cells via the CD44 receptor that stimulates the cells to migrate and to proliferate.
  • long GAG polymers i.e. > 10 2
  • oligosaccharides can be
  • HA polysaccharide plays structural roles in the eye, skin, and joint
  • HA-oligosaccharides composed of 10 to 14 sugars [HA10-14] have promise for inhibition of cancer cell growth and metastasis.
  • HA10-14 HA-oligosaccharides composed of 10 to 14 sugars [HA10-14] have promise for inhibition of cancer cell growth and metastasis.
  • mice injected with various invasive and virulent tumor cell lines were injected with various invasive and virulent tumor cell lines.
  • HA or HA-like oligosaccharides appear to serve as a
  • the preliminary mode of action of the HA-oligosaccharide sugars is thought to be mediated by binding or interacting with one of several important HA-binding proteins (probably CD44 or RHAM) in the mammalian body.
  • CD44 or RHAM important HA-binding proteins
  • multiple CD44 protein molecules in a cancer cell can bind simultaneously to a
  • each CD44 molecule individually binds a different HA molecule in a monovalent manner such that no dimerization/patching event occurs. Thus no activation signal is transmitted to the cell.
  • the optimal HA-sugar size is 10 to 14 sugars.
  • HA may be based more upon the size of HA currently available for testing rather
  • ⁇ 10 sugars are available according to the methodologies of the present invention, the optimal HA size or oligosaccharide composition may be found to
  • HA-oligosaccharides for therapeutics is that these sugar molecules are natural by-products that can occur in small amounts in the
  • Enhancement of wound-healing and resupplying cardiac oxygenation may be additional applications that harness the
  • Dendritic cells possess adjuvant activity in stimulating specific CD4 and CD8 T cell responses. Therefore, dendritic cells are targets in vaccine development strategies for the prevention and treatment of infections, allograft
  • Heparin interacts with many proteins in the body, but two extremely interesting classes are coagulation cascade proteins and growth factors.
  • Antithrombin III [ATIII] and certain other hemostasis proteins are 100,000-fold more potent inhibitors of blood clotting when complexed with heparin. Indeed,
  • heparin is so potent it must be used in a hospital setting and require careful monitoring in order to avoid hemorrhage. Newer, processed lower molecular
  • heparin is responsible for the ATIII-anticoagulant effect. But since heparin is a
  • the pentasaccharide can also be prepared in a conventional chemical synthesis involving ⁇ 50 to 60 steps. However, altering
  • VEGF vascular endothelial growth factor
  • HBEGF heparen-binding epidermal growth factor
  • FGF fibroblast growth factor
  • Chondroitin is the most abundant GAG in the human body, but all of its
  • Phenomenon such as neural cell
  • chondroitin or similar molecules are of utility in re-wiring
  • coagulation proteins such as heparin cofactor II.
  • glycosyltransferase enzymes that synthesize the alternating sugar repeat
  • UDP-sugar precursors and divalent metal cofactors e.g. magnesium, cobalt and/or manganese ion
  • divalent metal cofactors e.g. magnesium, cobalt and/or manganese ion
  • HexNAc GlcNAc or GalNAc.
  • n the degree of polymerization
  • GAG is polymerized by a single
  • the enzyme is called a synthase or co-polymerase.
  • the enzymes are: a HA synthase, or (pmHAS); a chondroitin synthase, or (pmCS); and two heparosan synthases, or (pmHS and PglA). To date, no keratan synthase from any source has been identified or reported in
  • pmHAS synthesis was disclosed and claimed. pmHAS is unique in comparison to all other existing HA synthases of Streptococcus bacteria, humans and an
  • recombinant pmHAS can elongate exogeneously-
  • HA chains e.g. 2-4 sugars
  • longer HA chains e.g. 3 to 150
  • the pmHAS synthase has been shown to add monosaccharides one at a time in a step-wise fashion to the growing chain.
  • the pmCS enzyme which is about 90% identical at the amino acids
  • heparosan is Beta4GlcUA-alpha4GlcNAc.
  • the pmHS and PglA enzymes were described and enabled in copending U.S. Serial No. 10/142,143.
  • pmHAS possesses two independent catalytic sites in one polypeptide. Mutants were created that transferred only GlcUA, and distinct mutants were also created that transferred only GlcNAc. These mutants cannot polymerize HA chains individually, but if the two types of mutants are mixed together in the
  • chondroitin synthase, pmCS has a similar sequence and similar two-domain
  • the heparosan synthases, pmHS and PglA also contain regions for the two active sites. Single action mutants have also been created for the chondroitin synthase, pmCS, and are described hereinafter in detail.
  • the Pasteurella GAG synthases are very specific glycosyltransferases with respect to the sugar transfer reaction; only the correct monosaccharide from
  • the authentic UDP-sugar is added onto acceptors.
  • the epimers or other closely structurally related precursor molecules e.g. UDP-glucose are not utilized.
  • the GAG synthases do, however, utilize certain heterologous acceptor sugars.
  • pmHAS will elongate short chondroitin acceptors with long HA chains.
  • pmHS will also add long heparosan chains onto HA acceptor
  • nucleic acid segment and “DNA segment” are referred to as used herein.
  • nucleic acid segment refers to a DNA segment which contains a
  • HAS Hyaluronate Synthase
  • CS Chondroitin Synthase
  • DNA segment includes DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • a DNA segment comprising an isolated or purified pmHAS or
  • pmCS or pmHS or PglA gene refers to a DNA segment including HAS or CS or
  • pmHAS or pmCS or pmHS or PglA forms the significant part of the coding region of the DNA segment, and that the DNA segment does
  • prokaryotic sources Due to certain advantages associated with the use of prokaryotic sources, one will likely realize the most advantages upon isolation of the HAS or CS or HS gene from the prokaryote P. multocida.
  • One such advantage is that, typically, eukaryotic genes may require significant post-transcriptional
  • prokaryotic enzyme gene is sought to be employed.
  • advantages include (a) the ease of isolation of a prokaryotic gene because of
  • DNA sequences in accordance with the present invention will be identical to each other.
  • the genetic control region may be native to the cell from which the gene was isolated, or may be native to the recombinant host cell, or may be an exaggerous segment that is compatible with and recognized by the transcriptional machinery of the selected recbominant host cell.
  • the nature of the control region employed will generally vary depending on the particular use (e.g., cloning host) envisioned.
  • the invention concerns isolated DNA segments
  • pmCS or pmHS or PglA gene that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO: 2, 4, 6, 8, 9, or 70,
  • HAS or CS or HS pepetides or peptide fragment thereof an amino acid sequence encoding HAS or CS or HS pepetides or peptide fragment thereof, and in particular to a HAS or CS or HS peptide or peptide
  • DNA segment or vector encodes a full length HAS or CS or
  • HS protein or is intended for use in expressing the HAS or CS or HS protein, preferred sequences are those which are essentially as set forth in SEQ ID NO: 1
  • Truncated pmHAS gene (such as, but not limited to, pmHAS 1"703 , SEQ ID NO: 1
  • Nucleic acid segments having HAS or CS or HS activity may be isolated by
  • SEQ ID NO:X means that the sequence substantially corresponds to a portion
  • SEQ ID NO:X has relatively few amino acids or codons encoding amino acids which are not identical to, or a biologically functional equivalent of, the amino acids or codons encoding amino acids of SEQ ID NO:X.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein, as a gene having a sequence essentially as set forth in
  • SEQ ID NO:X SEQ ID NO:X, and that is associated with the ability of prokaryotes to produce HA or a hyaluronic acid or chondroitin or heparin polymer in vitro or in vivo.
  • X refers to either SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 69, 70 or 71 or any additional sequences set forth herein, such as the
  • pmHAS 1"703 truncated or mutated versions of pmHAS 1"703 that are contained generally in SEQ ID NOS: 10-60.
  • nucleic acid segment i.e. encoding conserved or semi-conserved
  • a substituted nucleic acid segment may be highly identical and retain its enzymatic activity with regard to its unadulterated parent, and yet still fail to hybridize thereto. Additionally, the
  • present application discloses 4 enzymes and numerous mutants of these enzymes that still retain at least 50% of the enzymatic activity of the
  • the invention discloses nucleic acid segments encoding an enzymatically
  • nucleic acid segment that encodes a protein in accordance with SEQ ID NO:i or 3 or 5 or 7 or 71, respectively, further defined as a recombinant vector.
  • recombinant vector refers to a vector that has been modified
  • the recombinant vector may be further defined as an expression vector comprising a promoter operatively linked to said HAS- or CS- or HS- encoding nucleic acid segment.
  • a further preferred embodiment of the present invention is a host cell, made recombinant with a recombinant vector comprising an HAS or CS or HS
  • the preferred recombinant host cell may be a prokaryotic cell.
  • the recombinant host cell is an eukaryotic cell. As used
  • engineered or “recombinant” cell is intended to refer to a cell
  • engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene, such as a gene encoding HAS or CS or HS, has been introduced mechanically or by the hand of man. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene, such as a gene encoding HAS or CS or HS, has been introduced mechanically or by the hand of man. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene, such as a gene encoding HAS or CS or HS, has been introduced mechanically or by the hand of man. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene, such as a gene encoding HAS or CS or HS, has been introduced mechanically or by the hand of man. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene, such as a gene encoding HAS or CS or
  • Engineered cells are thus cells having a gene
  • genes positioned adjacent to a promoter associated or not naturally associated with the particular introduced gene include genes positioned adjacent to a promoter associated or not naturally associated with the particular introduced gene.
  • the HAS- or CS- or HS- encoding DNA In preferred embodiments, the HAS- or CS- or HS- encoding DNA
  • segments further include DNA sequences, known in the art functionally as origins of replication or "replicons", which allow replication of contiguous sequences by the particular host.
  • origins of replication or "replicons”
  • Such origins allow the preparation of extrachromosomally localized and replicating chimeric or hybrid segments or
  • the employed origin is one capable of replication in bacterial hosts suitable for biotechnology applications.
  • the employed origin may be desirable to alternatively or even
  • origins recognized by other host systems whose use is contemplated such as in a shuttle vector.
  • polyoma or bovine papilloma virus origins which may be employed for cloning or expression in a number of higher organisms, are well known to those of
  • the invention may thus be
  • HAS- or CS- or HS- coding gene sequence together with an appropriate replication origin and under the control of selected control regions.
  • HAS or CS or HS gene or cDNA may be used to obtain the HAS or CS or HS gene or cDNA, in light of the
  • polymerase chain reaction or RT-PCR produced DNA fragments may be obtained which contain full complements of genes or cDNAs from a number of sources, including other strains of Pasteurella
  • acids should encode a biologically functional equivalent HAS or CS or HS.
  • Typical useful vectors include plasmids and phages for use in prokaryotic organisms and even viral vectors for use in
  • eukaryotic organisms examples include pKK223-3, pSA3, recombinant
  • Gram-negative bacteria e.g. Bacillus, Lactococcus, or E. coli
  • the recombinant vector is employed to make the functional GAG synthase for in vitro use.
  • GRAS are benign and well studied organisms used in the production of certain foods and biotechnology products and are recognized as GRAS
  • Another procedure to further augment HAS or CS or HS gene copy number is the insertiori of multiple copies of the gene into the plasmid.
  • the chromosomal DNA-ligated vector is employed to transfect the host that is selected for clonal screening purposes such as E. coli, through the use of a vector that is capable of expressing the inserted DNA in the chosen host.
  • the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO: 1,3,5,7,69, or 71.
  • nucleic acid sequence substantially corresponds to a portion of SEQ ID NO: 1,3,5,7,69, or 71 and has
  • codon is used herein to refer to codons that encode the same amino acid, such
  • codons for arginine or serine refers to codons that encode biologically equivalent amino acids, as set forth in Table II.
  • additional residues such as additional N- or C-terminal amino acids or
  • terminal sequences particularly applies to nucleic acid sequences which may, for example
  • nucleotides of SEQ ID NO: 1,3,5,7,69, or 71 will be sequences which are "essentially as set forth" in SEQ ID NO: 1,3,5,7,69, or 71. Sequences which are essentially the
  • hybridization conditions will be well known to those of skill in the art and are clearly set forth hereinbelow. In a preferred embodiment, standard stringent hybridization conditions or less stringent hybridization conditions are utilized. The terms "standard stringent hybridization conditions," "moderately
  • stringency hybridization conditions are used herein, describe those conditions under which substantially complementary nucleic acid segments will form standard Watson-Crick base-pairing and thus “hybridize” to one another.
  • hybridization such as pH; temperature; salt concentration; the presence of agents, such as formamide and dimethyl sulfoxide; the length of the segments
  • the hybridizing portion of the hybridizing nucleic acids is typically at least about 14 nucleotides in length, and preferably between about 14 and about 100
  • the hybridizing portion of the hybridizing nucleic acid is at
  • At least about 60% e.g., at least about 80% or at least about 90%, identical to a portion or all of a nucleic acid sequence encoding a HAS or chondroitin or
  • heparin synthase or its complement such as SEQ ID NO: 1,3,5,7,69, or 71 or the complement thereof.
  • T m is the temperature at which a probe nucleic acid sequence dissociates from a target DNA. This melting temperature is used to
  • salt e.g., SSC, SSPE, or
  • the final wash temperature is decreased by about 5°C). In practice, the final wash temperature is decreased by about 5°C). In practice, the final wash temperature is decreased by about 5°C). In practice, the
  • T m can be between about 0.5°C and about 1.5°C per 1% mismatch.
  • Examples of standard stringent hybridization conditions include hybridizing at
  • Moderately stringent conditions include hybridizing as described above in 5xSSC ⁇ 5xDenhardt's solution 1% SDS washing in 3x SSC at 42°C.
  • parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Additional guidance regarding such conditions is readily available in the art, for
  • ATAGCG-3' is complementary to the sequence 5'-CGCTAT-3" because when the two sequences are aligned, each "T” is able to base-pair with an "A", which each "G” is able to base pair with a "C".
  • complementary sequences means nucleic acid sequences which are
  • nucleic acid segment of SEQ ID NO: 1,3,5,7, or 69, or 71 under standard stringent, moderately stringent, or less stringent hybridizing conditions.
  • nucleic acid segments of the present invention may be combined with other DNA
  • sequences such as promoters, polyadenylation signals, additional restriction
  • enzyme sites multiple cloning sites, epitope tags, polyhistidine regions, other coding segments, and the like, such that their overall length may vary
  • Recombinant vectors and isolated DNA segments may therefore variously include the HAS or CS or HS coding regions themselves,
  • coding regions bearing selected alterations or modifications in the basic coding region may encode larger polypeptides which nevertheless include HAS or CS or HS coding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acid sequences.
  • DNA segments of the present invention encompass DNA segments
  • polysaccharide must comprise at least 20-100 sugar units.
  • exogenous polymers have been previously available, but typically add only one sugar unit.
  • the unique enzymes described in the present invention e.g.
  • pmHAS, pmCS, pmHS, and PglA form polymers of at least 100-400 sugar units in length.
  • Pasteurella multocida bacteria normally make polymers similar to or identical
  • HA Type A bacteria
  • chondroitin Type F bacteria
  • heparosan unsulfated
  • the recombinant enzyme may be any suitable enzyme that catalyzes the oxidation of the recombinant enzyme.
  • the recombinant enzyme may be any suitable enzyme that catalyzes the oxidation of the recombinant enzyme.
  • the recombinant enzyme may be any suitable enzyme that catalyzes the oxidation of the recombinant enzyme.
  • pmHAS (SEQ ID NO: 2), a 972 amino acid residue protein from Pasteurella multocida, is made in a functional state in recombinant Escherichia coli.
  • the pmHAS gene is given in SEQ ID NO: 1.
  • Other functional derivatives of pmHAS for example an enzyme called pmHAS 1"703 (SEQ ID NO:9) and the pmHAS 1'703
  • the enzyme is in a "virgin" state since the
  • empty acceptor site can be occupied with foreign polymers.
  • the empty acceptor site can be occupied with foreign polymers.
  • recombinant enzyme may be incubated in a mixture comprising from about 10
  • Tris pH 7.2 from about 20 mM MnCI 2 , from about 0.1 to about 15 mM UDP-GlcUA, from about 0.1 to about 15 mM UDP-GlcNAc, and a suitable
  • acceptors can be any functional acceptor, such as a glycosaminoglycan acceptor
  • sugar acceptor for example, but not by limitation, short HA chains (two or more sugar units suc as HA 4 ) or short chondroitin sulfate chains (5 sugar
  • the length of the HA chain added onto the acceptor is controlled by
  • Immobilized acceptors such as beads or other solid objects with bound acceptor
  • oligosaccharides can also be extended by the pmHAS enzyme using UDP- sugars.
  • the pmHAS enzyme (or its derivatives) can be used to attach polysaccharide chains to any suitable acceptor molecule.
  • Type A P. multocida produces HA capsule [GlcUA-GlcNAc repeats] and possesses the pmHAS enzyme.
  • Type F P. multocida produces HA capsule [GlcUA-GlcNAc repeats] and possesses the pmHAS enzyme.
  • PmCS the P. multocida chondroitin synthase
  • pmHAS similar to pmHAS, one of ordinary skill in the art, given the present specification, is able to manipulate the pmCS in the same manner as that for
  • chimeric or hybrid polysaccharide materials can serve as a biocompatible molecular glue for cell/cell interactions in artificial tissues or organs and the
  • HA/chondroitin/heparin hybrid mimics natural proteoglycans that normally contain an additional protein intermediate between polymer chains.
  • polysaccharide devoid of such an intermediary protein, is desirous since molecules from animal sources are potentially immunogenic — the chimeric or
  • the recombinant polymers can be
  • glycosyltransferase remains in association with the nascent chain.
  • the nascent chain is covalentiy attached to the enzyme during its synthesis.
  • the nascent chain is covalentiy attached to the enzyme during its synthesis.
  • the nascent polymer chain is still covalentiy attached.
  • a nascent HA chain means for a nascent HA chain to be held at or near the active site.
  • pmHAS it appears ' that a HA-binding site exists near or at the sugar
  • oligosaccharides that vary in size and composition are used to discern the nature of the interaction between pmHAS and the sugar chain.
  • linkages from HA-derived oligosaccharides does not serve as an acceptor.
  • chondroitin [GlcUA-GalNAc repeat] does serve as an acceptor for pmHAS.
  • the cells were hyaluronidase-treated to give copious amounts of HA polysaccharide.
  • the cells were hyaluronidase-treated to
  • Both heparin and chondroitin are synthesized by the addition of sugar units to the nonreducing end of the polymer chain.
  • glycosyltransferases initiate chain elongation on at least primer
  • monosachharides More preferably tetrasaccharides such as xylose-galactose-
  • enzyme extracts transfer a single sugar to exogenously
  • heparin biosynthesis a single enzyme transfers both GlcUA and GlcNAc sugars to the glycosaminoglycan chain based on co-purification or expression studies.
  • heparosan indicates that a pair of proteins can transfer both sugars to the nonreducing end of acceptor molecules in vitro.
  • pmHAS adds single monosaccharides in a sequential fashion to the nonreducing termini of the nascent HA chain. Elongation of HA polymers containing hundreds of sugars has been demonstrated in vitro.
  • the glycosyltransferase may be stopped and started at different stages of synthesis of the heteropolysaccharide. In contrast, there is
  • glycosyltransferase enzymes that produce important homopolysaccharides such as chitin, cellulose, starch,
  • pmHAS 1"650 (SEQ. ID NO: 10) can only add
  • membrane preparations from recombinant E. coli containing a pmHAS protein had HA synthase activity as judged by
  • pmHAS 1"703 has been shown to add sugars onto a
  • chondroitin pentamer acceptor The pmHAS 1"703 and reagents were prepared in the same manner as shown in Fig.l, except that a chondroitin pentamer was used as the acceptor molecule. The results of this experiment are shown in TABLE III.
  • the pmHAS 1"703 can utilize molecules other than the
  • the HA polymerizing activity of recombinant pmHAS 1"703 is dependent on the simultaneous incubation with both UDP-sugar precursors and a Mn 2+ ion.
  • the level of incorporation is dependent on protein concentration, on HA
  • FIG. 2 two parallel reactions containing pmHAS 1"703 with even-numbered HA oligosaccharides (105 ⁇ g membrane protein/point with a mixture of HA
  • pmHAS 1"703 without oligosaccharide acceptor (630 ⁇ g protein/point; open circles) were compared.
  • the enzyme preparations were added to prewarmed
  • HA synthesized in the presence or the absence of HA oligosaccharides is sensitive to HA lyase (>95% destroyed) and has a molecular weight of ⁇ 1-
  • the native pmHAS 1"703 enzyme has an attached or bound nascent
  • recombinant enzyme on the other hand, lacks such a nascent HA chain since the E. coli host does not produce the UDP-GlcUA precursor needed to make HA
  • the exogenous HA-derived oligosaccharide has access to the active site of pmHAS 1"703 and can be elongated.
  • X cation and pH values used for each enzyme were: pmHAS 1"703 , Mn/7.2; Xenopous DG42, Mg/7.6; Group A streptococcal HasA, Mg/7.0), and enzyme (units/reaction listed).
  • pmHAS 1"703 Mn/7.2
  • Xenopous DG42 Mg/7.6
  • Group A streptococcal HasA Mg/7.0
  • FIG. 4 demonstrates that pmHAS 1"703
  • pmHAS extended an HA tetramer.
  • radiolabeled HA tetramer (HA4 8xl0 3 dpm 3 H) with a GlcUA at the nonreducing
  • the beginning of the analytical layer is marked by an arrow.
  • HA-derived oligosaccharides with either GlcUA or GlcNAc at the nonreducing terminus served as acceptors for pmHAS 1"703 (FIG. 5).
  • radiolabeled HA pentamer HA5, 5xl0 3 dpm 3 H
  • HA tetramer HA4, 25xl0 3
  • the pmHAS 1"703 enzyme transfers individual monosaccharides sequentially during a
  • Membrane preparations containing recombinant pmHAS (GenBank AF036004) (SEQ. ID NOS: 1 and 2) were isolated from E. coli SURE(pPmHAS).
  • HA oligosaccharides [(GlcNAc-GlcUA) n ] were generated by degradation of HA (from Group A Streptococcus) with either bovine testicular hyaluronidase Type
  • the negative ions produced by pulsed nitrogen laser irradiation were analyzed in linear mode (20 kV acceleration; Perceptive Voyager).
  • capsular polysaccharide is ⁇ 4GlcUA- ⁇ 4GlcNAc; this carbohydrate has the same
  • composition as HA but the glycosidic linkages between the monosaccharides are
  • the chitin-derived oligosaccharides, chitotetraose and chitopentaose are ⁇ 4GlcNAc polymer ⁇ made of 4 or 5 monosaccharides, respectively.
  • silica plates with application zones (Whatman) utilizing butanol/acetic
  • An anti-pmHAS monospecific antibody reagent has also been identified that routinely monitors the protein by Western blots or immunoassays; this
  • reagent can be used to normalize protein expression levels.
  • the DNA inserts encoding the enzyme sequence from interesting mutants picked up in screens can be subcloned and completely sequenced to verify and to identify the
  • membrane protein were created and are tabulated (with functionality) in Table V that produce proteins with altered physical properties (i.e. proteins that are more conducive to high-level expression and purification) and altered function
  • the truncation series was generated and tested for activity. All proteins were made at the expected molecular weight, but not all proteins were active.
  • pmHAS 1 80-kDa protein
  • pmHAS 1"703 was purified by sequential chromatography steps shown in FIG. 6.
  • FIG. 6 a soluble, active form of the
  • HA synthase was constructed with molecular biological techniques.
  • the recombinant enzyme from E. coli was purified by conventional chromatography with yields of up to 20 mg/liter of cell culture.
  • FIG. 6 is a stained electrophoretic gel loaded with samples of pmHAS 1"703 (marked with an arrow) during different stages of chromatography.
  • This catalyst (and improved mutant versions) can be used to prepare HA coatings on artificial surfaces or HA extensions on suitable acceptor molecules.
  • the pmHAS 1"703 is highly active and at least 95% pure as assessed by
  • Synthetic DNA oligonucleotides and multiple rounds of extension with Pfu DNA polymerase were used to add mutations to the coding region using the Quick-Change system from Stratagene.
  • mutant enzymes are useful for adding on a single GlcNAc or a single GlcUA
  • FIG. 8 is a graphical representation of an experiment where combining
  • Extracts of the mutants were used for all three kinds of assays: for HA polymer production, for GlcUA-Tase activity and for GlcNAc-Tase activity.

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Abstract

La présente invention concerne une méthodologie de greffe polymère par une synthase de polysaccharide et, plus particulièrement, la greffe polymère effectuée à l'aide de synthases de hyaluronate ou de chondroïtine ou d'héparine/héparosane issues de Pasteurella multocida, qu'on effectue afin de créer une variété d'oligosaccharides de glycosaminoglycane ayant une structure de sucre naturelle ou chimérique ou bien hybride.
PCT/US2002/022386 2001-07-13 2002-07-12 Polymeres de glycosaminoglycane et procedes de preparation et d'utilisation de ces derniers WO2003029261A2 (fr)

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US8580290B2 (en) 2001-05-08 2013-11-12 The Board Of Regents Of The University Of Oklahoma Heparosan-based biomaterials and coatings and methods of production and use thereof
US9687559B2 (en) 2008-03-19 2017-06-27 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9925209B2 (en) 2008-03-19 2018-03-27 The Board Of Regents Of The University Of Oklahoma Heparosan-polypeptide and heparosan-polynucleotide drug conjugates and methods of making and using same
US8697398B2 (en) 2010-03-01 2014-04-15 Dsm Ip Assets B.V. Compositions and methods for bacterial production of chondroitin
WO2013149161A1 (fr) 2012-03-30 2013-10-03 Deangelis Paul L Polymères d'héparosane de poids moléculaire élevé et procédés de production et d'utilisation de ceux-ci
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Class et al. Patent application title: Production of Defined Monodisperse Heparosan Polymers and Unnatural Polymers with Polysaccharide Synthases Inventors: Paul L. Deangelis (Edmond, OK, US) Paul L. Deangelis (Edmond, OK, US) Alison Sismey-Ragatz (Cassville, WI, US)

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