WO2007083274A1 - Sulfate de dermatane épimérase essentiellement pure - Google Patents

Sulfate de dermatane épimérase essentiellement pure Download PDF

Info

Publication number
WO2007083274A1
WO2007083274A1 PCT/IB2007/050162 IB2007050162W WO2007083274A1 WO 2007083274 A1 WO2007083274 A1 WO 2007083274A1 IB 2007050162 W IB2007050162 W IB 2007050162W WO 2007083274 A1 WO2007083274 A1 WO 2007083274A1
Authority
WO
WIPO (PCT)
Prior art keywords
epimerase
seq
dermatan sulfate
activity
substantially pure
Prior art date
Application number
PCT/IB2007/050162
Other languages
English (en)
Inventor
Ulf Lindahl
Jin-Ping Li
Anders MALMSTRÖM
Marco Maccarana
Original Assignee
Polysackaridforskning I Uppsala Ab
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
Application filed by Polysackaridforskning I Uppsala Ab filed Critical Polysackaridforskning I Uppsala Ab
Publication of WO2007083274A1 publication Critical patent/WO2007083274A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)

Definitions

  • the present invention relates to substantially pure chondroitin glu- curonate C-5 epimerase or dermatan sulfate (DS) epimerase. Also disclosed is the use of such substantially pure DS epimerase in synthetic processing of defructolsylated E. coli K4 polysaccharide (dK4) to obtain epimerized dermatan sulfate.
  • the invention further relates to the gene encoding DS epimerase, and to the use of DS epimerase encoding DNA for the recombinant production of DS epimerase.
  • Proteoglycans are complex molecules comprising, in addition to the core protein, glycosaminoglycan (GAG) chains, which are important for their biological functions.
  • GAG glycosaminoglycan
  • DS proteoglycans carry linear polysaccharides of alternating N-acetylgalactosamine (GaINAc) and L-iduronic acid (IdoA), or D-glucuronic acid (GIcA).
  • DS The biological functions of DS span from basic cellular processes such as cell division in C. elegans to cell type specific roles such as the neurite outgrowth promoting activity of rare structures present in DS.
  • Other important functions of DS are the binding to several growth factors and the participation in processes such as coagulation and wound healing.
  • coagulation DS affects both antithrombin and protein C, and it is the main GAG found in wound fluid where it promotes FGF2 activity and fibroblast proliferation.
  • migration is mediated by the DS proteoglycans biglycan and decorin. Highly sulfated DS structures inhibit fibroblast proliferation. Finally DS stimulates ICAM-1 production.
  • a therapeutic potential of DS proteoglycans is thus undisputable, and there is a recognized need for highly purified, preferably (semi)- synthetically produced DS chains.
  • the biosynthesis of DS is initiated by the addition of a xylose to a serine residue in the core protein by a xylosyltransferase, the only step occurring in the ER.
  • a xylosyltransferase the only step occurring in the ER.
  • two galactose residues and one glucuronic acid are sequentially added to generate the linkage tetrasaccharide region, common for heparan sulfate (HS) and DS.
  • HS heparan sulfate
  • the subsequent alternating addition of GaINAc and GIcA by a family of glycosyltransferases generates the full-length chondroitin chain.
  • the chain undergoes to various extent modifications such as epimerization of GIcA to iduronic acid and O-sulfation at position 2 of GlcA/ldoA and at position 4 and 6 of GaINAc.
  • Uronysyl-2-O-sulfotransferase (ST) sulfates carbon position 2 of both GIcA and IdoA, and C4ST1 and D4ST1 sulfate position 4 of GalNac flanked by two GIcA or IdoA residues, respectively.
  • C4ST2 acts on both structures, while the specificity of C4ST3 is still unclear .
  • IdoA from GIcA is catalyzed at polymer level by the chondroitin glucuronate C5-epimerase - DS-epimerase - the only enzyme in the biosynthetic process which so far has not been cloned.
  • This reaction converts GIcA into IdoA, which endows conformation flexibility to the chain (2), believed to be important for protein interactions.
  • This enzyme uses non- sulfated chondroitin as substrate (3), but to generate a DS chain containing more than 15% of IdoA, concurrent 4-O-sulfation needs to occur (4).
  • IdoA residues are indeed found next to 4-O-sulfated GaINAc.
  • the detailed mechanisms of 4-O-STs and uronosyl 2-O-sulfotransferase in conjunction with the epimerase action remain to be elucidated (5).
  • the ratio between GIcA and IdoA not only differs between tissues but also between different DS proteoglycans and it seems to be core protein dependent. For instance, in human lung fibroblasts, versican has a very low IdoA content compared to decorin and biglycan. IdoAs are usually found adja- cent to each other in DS, which therefore results in a copolymeric structure with alternating IdoA and GIcA blocks. This closely resembles the iduronic acid clustering in heparan sulfate. The formation of IdoA blocks might indicate a processive mode of action of the DS-epimerase.
  • the present invention relates to substantially pure dermatan sulfate epimerase, especially to dermatan sulfate epimerase purified from bovine spleen.
  • the present invention also relates to nucleic acids encoding dermatan sulfate epimerase, including bovine and human DS epimerase.
  • the present invention furthermore relates to vector comprising nucleic acids according to the present invention and to host cells comprising such vectors.
  • the present invention furthermore relates to methods for producing dermatan suhate epimerase, comprising cultivating a host cell according to the present invention and recovering said DS epimerase from cellular lysate.
  • the present invention further relates to the use of nucleic acids ac- cording to the present invention for the production of recombinant DS epimerase.
  • the present invention also relates to the use of substantially pure DS epimerase for producing dermatan sulfate, especially where E. coli K4 polysaccharide is used as starting material.
  • Figure 1A presents the chromatographic purification of DS epime- rase on Superose 12 (step 5 in Table I). Arrows indicate elution position of molecular weight markers.
  • Figure 1 B shows the SDS-PAGE separation of purified DS epime- rase. Lane 1 : the 43,000 -fold most purified preparative sample (see Table I). Lane 2: the 600,000 -fold purified analytical sample (see Step 7 of Example 1 ) The arrow indicates the predominant 89 kDa band.
  • Figure 1C shows the alignment of human SART2 (SwissProt Q9UL01 ) with bovine SCAFFOLD270174.1.
  • the unique MS/MS peptides (Table II) matched to the sequences are underlined.
  • FIG. 2A and B Expression of SART2/DS epimerase.
  • Cells were transiently transfected with empty pcDNA 3.1/His expression vector (empty bars) or with a vector containing SART2/DS epimerase in frame with C- terminal His tag (filled bars).
  • Figures 3A to 3C show the results of an analysis of the products of an in vitro epimerase reaction.
  • FIG. 3A Size analysis of intact dK4: 3H-dK4 incubated with control ( ) or DS epimerase overexpressing ( ⁇ ) lysate; 14C-dK4 incubated with control (o) or DS epimerase overexpressing (•) lysate.
  • FIG. 3B Size analysis of chondroitinase AC-I split products. dK4 not incubated with enzyme ( ⁇ ), or incubated with control (o) or DS epimerase overexpressing (•) lysate. Elution positions of di-, tetra- and hexasacchahdes derived from dK4 are indicated by arrows.
  • Figure 3C Paper chromatography of disaccharides. 14 C-dK4 incubated with control (o) or DS epimerase overexpressing (•) lysate.
  • Figure 5A shows the effect of pH, obtained by overlapping 10 mM acetate (•) and 10 mM MES ( ⁇ ) buffers
  • Figure 5B shows the effect of the divalent cation MnCb (•), CaCb ( ⁇ ), or Mg Cb (V)
  • Figure 5C shows the effect of the monovalent cation NaCI ( ⁇ ) or KCI (•).
  • Figure 6 shows the rat tissue distribution of epimerase activity.
  • a purification protocol according to the present invention it was finally possible to purify DS epimerase approximately 43, 000-fold from spleen microsomes.
  • This protocol enabled the inventors to prepare trypsinized peptides and to run a database comparison on the peptide sequences. It was then surprisingly found that DS epimerase is encoded by a gene earlier sequenced and presented as SART-2. However, no specific function has ever been assigned to this protein.
  • a purification protocol according to the present invention it was possible to purify DS epimerase -600,000-fold from spleen microsomes. Silver stained SDS-PAGE showed a 89 kDa band as prominent band. The 89 kDa band, analyzed by mass spectrometry in the -43,000-fold purified sample, contained SART2/DS epimerase. It is thus an object of the present invention to provide substantially pure DS epimerase.
  • Microsomal preparation was first applied to Red Sepharose gel, allowing a 10-fold purification and an excellent recovery.
  • ConA Sepharose column was very efficient in the removal of contaminants, but allowed only 30-40% recovery, giving a 7-fold purification.
  • dK4 Sepharose gave a consistent 3-fold purification and high recovery.
  • concentration the material was applied to a Superose 12 column. Most of the protein came out as high-molecular-weight complexes, while the epimerase frac- tion was much more homogeneous in size, eluting 4-fold purified with the peak of activity at the position of a 67-kDa marker ( Figure 1A).
  • the size fractionation was refined by re-applying the material to the same column, which showed a sharp activity peak with two fractions containing 80% of the eluted activity, again with a 4-fold purification.
  • the most active frac- tion was incubated in a batch mode with a tiny amount of Red Sepharose gel. Most of the activity was recovered and was 14 times more purified. The complexity of this sample, purified altogether approximately 43,000 times, was checked on SDS-PAGE ( Figure 1 B, Lane 1 ).
  • the essen- tially pure DS epimerase is of bovine origin.
  • Said bovine DS epimerase according to the present invention is preferably provided at least 40,000 -fold purified, more preferably at least 500,000-fold purified, as compared to the activity of DS epimerase in bovine spleen microsomal preparation.
  • FOLD172907.1 (2 peptides; prolylcarboxypeptidase).
  • AIYDIVHR (SEQ ID NO 2) h 9865423 097 26 016 12/14
  • AIYDIVHR (SEQ ID NO 3) b 9865423 098 25 027 13/14
  • GEGVGAYNPQLHLR (SEQ ID NO 6) b 1510777 1 33 049 19/26
  • MAAQPSWLVK (SEQ ID NO 9) h 1130603 098 29 027 16/18
  • WAAVEKNGWFIR (SEQ ID NO 17) b 150089 1 21 021 20/26
  • YTFFNNVLMFSPAASK (SEQ ID NO 18) b 18369 093 28 023 15/30 a: All peptides were derived from consensus trypsin cleavage sites.
  • c Peptide prophet probability score (35).
  • f Number of matched ions over observed ions.
  • said substantially pure DS epimerase is a bovine DS epimerase, encoded by a DNA sequence according to SEQ ID NO.21, and comprising the deduced amino acid sequences as set forth in SEQ ID NO:s 23 and 24.
  • said DS epimerase is a human DS epimerase encoded by a DNA sequence according to SEQ ID NO. 22, and comprising the amino acid sequence of SEQ ID NO: 25.
  • human DS epimerase gene consists of 6 exons in location 6q22 and HS epimerase of 3 exons in location 15q23 (41 ;42).
  • DS epimerase seems to be highly conserved between different species: compared with the human sequence, bovine (XP_591812.2, predicted sequence) has 91 % amino acid identity, mouse (NP_766096.1 ) 93%, chicken (XP_419777.1 ; predicted sequence) 86%, and zebrafish (CAI20604.1 ; predicted sequence) 65%.
  • the 958aa-long human DS epimerase has one putative transmembrane region at its N-terminus (aa 8-30), and two at its C-terminus [aa 901 -923 and 931 -952; see Fig. 1 C].
  • SART2/DS epimerase expression A full length SART2 cDNA was subcloned into an expression vector in frame with C-terminal His tag, and transiently transfected into three cells lines.
  • the transfection data unambiguously indicated that SART2 DNA codes for a protein that can act on the C5-3H-labeled substrate, liberating a volatile radioactive component, which is measured in the assay. It is thus a further object of the present invention to provide a method of producing recombinant DS epimerase, comprising transfecting a cell line with a vector comprising a cDNA sequence encoding for DS epimerase, cultivating said transfected cell lines, and recovering DS epimerase from cellular lysates. Effect of DS epimerase on dK4. We wanted to ascertain the structure of the product of the enzyme.
  • the [1 - 14 C]-labeled polysaccharide product was recovered and idu- ronic acid content was evaluated with two different strategies.
  • the polysaccharide was depolymerized by chondroitinase AC-I, which cleaves be- tween GaINAc and GIcA, and the mixture was applied to a Superdex Peptide column (Fig. 3B and inset).
  • Incubation with a mock-transfected lysate gave rise to 0.2%, 1 %, and 99% of radioactivity eluting in the position of hexa-, tetra-, and disacchahdes.
  • the corresponding values for epimerase-transfected cells were 2.5%, 12%, and 85%. According to the previous analysis, the amount of IdoA was 0.6% of total HexA, as a result of incubation with mock-transfected cellular lysate, and 8% when overexpressing lysate was used.
  • DS chains produced in cells by DS epimerase 293HEK cells were grown in the presence of 35 SO4, and the isolated DS chains were subjected to chondroitinase ABC, B, and AC-I digestion. 35 S-labeled chains were quantitatively degraded to disaccharides by chondroitinase ABC (data not shown), indicating that no radioactive contaminant was present in the DS preparation. Chondroitinase B, which cleaves between GaINAc and IdoA, pro- prised a profile on size permeation chromatography (Fig.
  • the present invention also provides a method of preparing epimerized E.coli K4 polysaccharides, by fermenting bacteria in a suitable medium, lysing said bacteria and extracting capsular polysaccharide by ion-exchange chromatography on a DEAE-column as described in (7).
  • the extracted polysaccharides are thereafter defructosy- lated as described in (7), whereafter the epimehzation step is performed as described above using substantially pure DS epimerase, followed by chemical sulfation as described in (10;11 ).
  • DS epimerase extensively purified from bovine spleen according to the present invention, was assayed, varying each time the parameter under study (Fig. 5).
  • DS epimerase activity has a pH optimum at pH 5-5.5 using dK4 as substrate and pH 6.5 using dermatan as substrate (3).
  • Enzymatic activity was also measured in different rat organs, from which total detergent-extracted lysates was prepared (Fig. 6).
  • Spleen had the highest specific activity, followed by stomach, uterus, and ovary, which had about one third of the activity of spleen, and kidney, lung, liver, which had about one tenth of the activity of spleen.
  • Skeletal muscle, heart, and brain contained little but detectable activity, whereas serum contained no detectable activity.
  • the enzyme is therefore ubiquitously active in tissues, but with big dif- ferences. In particular, brain contained approx. 20 times less specific activity than the mean of 10 tissues.
  • the quantitative comparison of specific activity correlated well with the ubiquitous mRNA expression of a 4.0-Kb SART2 transcript (12), with highest expression in kidney and ovary and lowest expression in colon, thymus, and brain.
  • Me- tabolically 3 H- or 14 C-labeled K4 was prepared by incorporating D-[5- 3 H]glucose or D-[1 - 14 C]glucose into the medium of K4 producing E.coli strain culture, as described in (7).
  • the resulting labeled K4 polysaccharides were defructosylated (dK4).
  • Incubations were performed in final 100 ⁇ l of 0.8X desalting buffer, 0.5 mg BSA carrier, 2 mM MnCI2, 0.5% NP40, and 30,000 dpm [ 5 -3H]-dK4 or [1 - 14 C]-dK4 (corresponding to approx 200 ⁇ M HexA).
  • the enzymatic reaction was performed at 37°C for 2-14 h, stopped by boiling for 5 min, and spun down 5 min at 10,000 rpm.
  • [5- 3 H]-dK4 substrate was used, because it releases tritiated water upon enzymatic attack, 90 ⁇ l of the incubations were transferred to a distillation tube containing 200 ⁇ l of water. After distillation (13), 200 ⁇ l of the distilled solution was counted.
  • the assay is linear to 3000 dpm of released tritiated water.
  • K4 polysaccharide is prepared as in (7). After defructosylation (15), it was coupled to AH-Sepharose, by a car- bodiimide procedure (16).
  • Bovine spleen was obtained fresh from the local slaughterhouse and processed immediately. All procedures were carried out at 4°C and all buffers contained 1 mM DTT as reducing agent.
  • Step 1 Microsomal preparation and extraction.
  • Day 1 Three spleens were freed from surrounding fat tissue, cut into 1 - to 2-cm cubes, washed twice with cold distilled water and placed in homogenization buffer (20 mM MES pH 6.5, 250 mM sucrose, 5 mM EDTA, protease inhibitors as follows: 1 mM PMSF, 1 ⁇ g/ml each pepstatin, aprotinin, leupeptin). Batches of 350 g of tissue were first homogenized without any added buffer in a food processor and were then re-homogenized three times after step-additions of 350, 500, and 500 ml of buffer.
  • homogenization buffer 20 mM MES pH 6.5, 250 mM sucrose, 5 mM EDTA, protease inhibitors as follows: 1 mM PMSF, 1 ⁇ g/ml each pepstatin, aprotinin, leupeptin.
  • the homogenate was centrifuged for 15 min at 800Og.
  • the resulting supernatant was centrifuged at 38,40Og for 45 min.
  • the 38,40Og pellet was extracted with 90 ml of solubilization buffer (20 mM MES pH 6.5, 150 mM NaCI, 1 mM EDTA, 1 % glycerol, 1 % Triton X-100, protease inhibitors).
  • solubilization buffer (20 mM MES pH 6.5, 150 mM NaCI, 1 mM EDTA, 1 % glycerol, 1 % Triton X-100, protease inhibitors).
  • the combined extracted material from 11 such preparations in all corresponding to 3.9 kg of tissue, was subjected to two strokes with a 200-ml Potter device and centrifuged at 38.400 g for 45 min. The supernatant was collected.
  • Step 3 ConA Sepharose.
  • the eluted material from Step 2 was adjusted to 1 mM MgCI 2 , 1 mM CaCI 2 , 0.1 % Triton X-100 and applied to a column of ConA Sepharose (2.6 x 6 cm; gel not re-utilized) run at 2 ml/min.
  • the gel was washed with 30 bed volumes of 10 mM MES pH 6.5, 0.1 mM EDTA, 10% glycerol, 1 mM MnCI 2 , 1 mM CaCI 2 , 0.1 % Triton X-100 (Buffer B)/ 0.5 M NaCI, and then with 6 bed volumes of buffer B/20 mM NaCI.
  • Step 4 dK4 Sepharose.
  • the eluate from Step 3 was applied to a dK4- Sepharose column (2.6 x 3 cm) at 2 ml/min.
  • the column was washed with 10 bed volumes of buffer A/20 mM NaCI/10 mM CHAPS, further washed with 10 bed volumes of buffer A/20 mM NaCI, and finally eluted with buffer A/150 mM NaCI.
  • Fractions with epimerase activity were pooled, dialyzed versus buffer A/1 OmM NaCI, and concentrated by applying them to a Mono Q 5/5 column, run at 0.5 ml/min. After application, the column was inverted, and eluted at a flow rate of 0.1 ml/min with buffer A/1 M NaCI.
  • Step 5 Superose 12.
  • the 1 -ml concentrated material from Step 4 was injected to a Superose 12 column, that was subsequently eluted at 0.05 ml/min with buffer A/150 mM NaCI. Fractions of approx. 0.3 ml were collected (Fig. 1A) and analyzed for epimerase activity. Active fractions were pooled, diluted with buffer A to a final NaCI cone, of 20 mM, and concentrated on a Mono Q PC 1.6/5 column, operated as described above.
  • Step 6 Superose 12. The 0.2-ml concentrated material from Step 5 was injected to a second Superose 12 column, operated as above.
  • Step 7 Red-Sepharose.
  • the most active fraction from the previous step was diluted with buffer A to 100 mM NaCI, CHAPS was added to final 10 mM cone, and the sample was batch-incubated with 50 ⁇ l of fresh Red Sepha- rose gel. After incubation for 2 h, the gel was washed 5 times with 500 ⁇ l of buffer A/100 mM NaCI/10 mM CHAPS, further washed 5 times with 1 ml of the same solution without CHAPS, and finally eluted with 10 x 150 ⁇ l buffer A/2M NaCI.
  • the -0.6 million-fold purified analytical sample shown in Fig. 1 B lane 2 was prepared as above, with one modification; an initial elution step with buffer A/1 M NaCI preceded the final elution with buffer A/2M NaCI.
  • the final eluate contained -10% of the epimerase activity incubated with the Red Sepharose gel.
  • Tryptic peptide preparation Material from the last purification step was precipitated with TCA, and the pellet was resuspended in reducing Laemli buffer. SDS-PAGE was carried out on NuPage pre-made 10% acrylamide gels (Invitrogen) that were stained with Brilliant Blue G Colloidal staining solution (Sigma). Gel bands of interest were cut out and the proteins were digested with trypsin (Promega) as described (17).
  • ESI-based LC-MS/MS analyses were carried out using an Agilent 1100 series instrumentation (Agilent Technologies, Paolo Alto, CA, USA) on a 75- ⁇ m x 10.5-cm fused silica microcapillary reversed phase column (5 ⁇ m Magic C18 beads, Michrom Bioresources). The column was eluted at 200 nl/min using a gradient of 5-35% acetonithle in 0.1 % formic acid, over a 50-min period as described (18).
  • LC-MS/MS was performed using an LTQ iontrap (Thermofinnigan, San Jose, CA, USA) with an electrospray voltage of 2 kV.
  • the instrument was set up to perform one MS scan (400-1600 Da) followed by three MS/MS analyses in a data-dependent mode with an intensity threshold of 15.000 counts.
  • the repeat count was set to 3, the repeat duration to 30 s, the exclusion duration was 60 s and the exclusion list size was 100.
  • Database search was carried out using SEQUEST (19) and the search results were further analyzed by INTERACT as previously described (8;9).
  • SART2 human cDNA clone (IMAGE no. 5272885) was obtained from RZPD, Germany, and subcloned into pcDNA 3.1/myc-His vector (Invitro- gen) using Xhol and Agel restriction sites introduced by PCR using primers 5'- GATCCTCGAGATGAGGACTCACACACGGGG-3' (SEQ ID NO 19) and 5'- GATCACCGGTACACTGTGATTGGGAACAAGA-3' (SEQ ID NO 20), respec- tively. The insert was confirmed by sequencing. Ligation into the expression vector resulted in a construct with SART2 in frame with a C-terminal 6XHis tag.
  • CHO-K1 cells maintained in F12-K medium/10%FBS, HFL-1 cells and 293HEK cells, both maintained in MEM/10% FBS, were grown in 6-well plates and transiently transfected with pcDNA-His, or pcDNA SART2-His plasmid, using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer's instruction. After 48 h, cells were washed with PBS, and lysed in 20 mM MES pH 6.5, 150 mM NaCI, 10% glycerol, 2 mM DTT, 1 mM EDTA, 1 % Triton X- 100, protease inhibitors.
  • cell lysate was centhfuged for 1 h at 20.000 g, and 200 ⁇ l of the supernatant was desalted (without carrier BSA added prior to desalting), followed by determination of protein content and enzyme activity.
  • samples were subjected to hydrazinolysis (64% hydrazine/36% H2O/1 % hydrazine sulfate) in a boiling bath for 16 h, the polysaccharide reiso- lated from PD-10 column run in water, and then treated with HNO2 at pH 3.9 (20).
  • the resultant disaccharides representing 64% of the total radioactivity, were reduced with NaBH 4 and recovered by gel chromatography on Superdex Peptide column, run at 0.3 ml/min in 0.2 M NH 4 HCOs.
  • the labeled disaccharides were separated by paper chromatography according to (7).
  • Example 7 Cellular CS/DS chains labeling, isolation, and lyase digestion
  • 293HEK cells grown in 75-cm 2 flask were transiently transfected, as described in Example 4, with pcDNA-His, or pcDNA SART2-His plasmid. After transfection, cell were grown for 24 h in ordinary medium, washed once in sulfur-deprived MEM, grown 2 h in sulfur-deprived MEM, 10% FBS, and finally 35 SO 4 was added to final 100 ⁇ Ci/ml. Cells were grown for an additional 24 h. At the end of the labeling period, medium was collected and applied to a 2-ml DE52 column, equilibrated in 50 mM acetate pH 5.5, 0.1 % Triton, 6 M urea.
  • the column was washed with 30 bed volume of equilibration buffer, further washed with 5 volumes of water, 5 volumes of 0.2 M NH 4 HCOs, and finally eluted with 10 volumes of 2 M NH 4 HCOs.
  • Samples were lyophilized, 10 ⁇ g of cold DS was added, subjected to ⁇ -elimination, and GAG chains were re- isolated using DE52 column, operated as described above. The GAG chains were deaminated at pH 1.5 (20), and intact CS/DS chains were recovered after gel filtration.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne une sulfate de dermatane épimérase essentiellement pure, des séquences d'acides nucléiques codant pour la sulfate de dermatane épimérase bovine et la sulfate de dermatane épimérase humaine, des vecteurs comprenant de telles séquences d'acides nucléiques ; des cellules hôtes comprenant lesdits vecteurs ainsi que des méthodes d'obtention de sulfate de dermatane épimérase essentiellement pure. En outre, la présente invention concerne l'emploi d'une SD épimérase essentiellement pure dans l'obtention de sulfate de dermatane.
PCT/IB2007/050162 2006-01-19 2007-01-18 Sulfate de dermatane épimérase essentiellement pure WO2007083274A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI20065035 2006-01-19
FI20065035A FI20065035A (fi) 2006-01-19 2006-01-19 Olennaisesti puhdas entsyymi

Publications (1)

Publication Number Publication Date
WO2007083274A1 true WO2007083274A1 (fr) 2007-07-26

Family

ID=35883933

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2007/050162 WO2007083274A1 (fr) 2006-01-19 2007-01-18 Sulfate de dermatane épimérase essentiellement pure

Country Status (2)

Country Link
FI (1) FI20065035A (fr)
WO (1) WO2007083274A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017106782A1 (fr) * 2015-12-18 2017-06-22 Tega Therapeutics, Inc. Compositions de glycosaminoglycane cellulaire et leurs procédés de préparation et d'utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996014425A1 (fr) * 1994-11-04 1996-05-17 Inalco S.P.A. Polysaccharides a teneur elevee en acide iduronique
JPH11318455A (ja) * 1998-05-08 1999-11-24 Kyogo Ito ヒト癌退縮抗原タンパク質
WO2004024097A2 (fr) * 2002-09-16 2004-03-25 Genentech, Inc. Compositions et methodes de traitement de maladies de nature immune

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996014425A1 (fr) * 1994-11-04 1996-05-17 Inalco S.P.A. Polysaccharides a teneur elevee en acide iduronique
JPH11318455A (ja) * 1998-05-08 1999-11-24 Kyogo Ito ヒト癌退縮抗原タンパク質
WO2004024097A2 (fr) * 2002-09-16 2004-03-25 Genentech, Inc. Compositions et methodes de traitement de maladies de nature immune

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
DATABASE EM_HUM [online] NAKAO MASANOBU ET AL.: "Identification of a Gene Coding for a New Squamous Cell Carcinoma Antigen Recognized by the CTL1", XP003012354, Database accession no. (AF098066) *
DATABASE GSP [online] XP003012355, Database accession no. (ADL83102) *
DATABASE GSP [online] XP003012387, Database accession no. (AAY51120) *
DATABASE WPI Week 200008, Derwent World Patents Index; AN 2000-090523, XP003012353 *
EKLUND F. ET AL.: "Dermatan Is a Better Substrate for 4-O-Sulfation Than Chondroitin: Implications in the Generation of 4-O-Sulfated,L-Iduronate-Rich Galactosaminoglycans", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol. 383, no. 2, November 2000 (2000-11-01), pages 171 - 177, XP003012356 *
HANNESSON H.H. ET AL.: "Biosynthesis of dermatan sulphate", BIOCHEM. J., vol. 313, 1996, pages 589 - 596, XP003012357 *
J. IMMUNOL., vol. 164, 2000, pages 2565 - 2574 *
MACCARANA M. ET AL.: "Biosynthesis of Dermatan Sulfate", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, no. 17, 28 April 2006 (2006-04-28), pages 11560 - 11568, XP003012359 *
SEIDLER D.G. ET AL.: "Core Protein Dependence of Epimerization of Glucuronosyl Residues in Galactosaminoglycans", J. BIOL. CHEM., vol. 277, no. 44, 1 November 2002 (2002-11-01), pages 42409 - 42416, XP003012358 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017106782A1 (fr) * 2015-12-18 2017-06-22 Tega Therapeutics, Inc. Compositions de glycosaminoglycane cellulaire et leurs procédés de préparation et d'utilisation
US11549000B2 (en) 2015-12-18 2023-01-10 Tega Therapeutics, Inc. Cellular glycosaminoglycan compositions and methods of making and using

Also Published As

Publication number Publication date
FI20065035A (fi) 2007-07-20
FI20065035A0 (fi) 2006-01-19

Similar Documents

Publication Publication Date Title
Maccarana et al. Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5-epimerase is identical to SART2
JP6670235B2 (ja) 可逆的ヘパリン分子、その製法及びその使用方法
Ehrhardt et al. In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation
EP2655421B1 (fr) Synthèse chimio-enzymatique d'héparines structurellement homogènes et de masse moléculaire ultra-faible
Yang et al. Depolymerized glycosaminoglycan and its anticoagulant activities from sea cucumber Apostichopus japonicus
JP3818676B2 (ja) ヘパラン硫酸６−ｏ−硫酸基転移酵素
Rhyner et al. Characterization of the human calmodulin-like protein expressed in Escherichia coli
WO2009014715A2 (fr) Synthèse enzymatique de polysaccharides sulfatés sans résidus d'acide iduronique
Zueva et al. Expression and biochemical characterization of two recombinant fucoidanases from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T
US20230340552A1 (en) Methods for synthesizing anticoagulant polysaccharides
US11473068B2 (en) Engineered aryl sulfate-dependent enzymes
CN108531470B (zh) 一种硫酸岩藻多糖裂解酶tflfm及其制备方法和应用
WO2004017910A2 (fr) Synthese totale de l'heparine
JPH0928374A (ja) ヘパラン硫酸 ２−ｏ−硫酸基転移酵素
US20080207895A1 (en) Methods for synthesizing polysaccharides
Wang et al. High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme
WO2007083274A1 (fr) Sulfate de dermatane épimérase essentiellement pure
Hasan et al. The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms
JP2009060888A (ja) ヒアルロン酸加水分解酵素及び該酵素を用いた低分子化ヒアルロン酸の製造方法
US20230151339A1 (en) Engineered aryl sulfate-dependent enzymes
EP4182452A1 (fr) Procédés de synthèse de sulfate d'héparane non anticoagulant
KR20220032086A (ko) 항응고제 다당류의 합성 방법
WO2023081336A2 (fr) Procédés de synthèse chimioenzymatique d'héparine de bas poids moléculaire à partir d'héparosane de bas poids moléculaire
WO2023118313A1 (fr) Héparane sulfate amélioré et ses procédés de fabrication
Ito et al. The linkage of teleost skin keratan sulfate to protein

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07700622

Country of ref document: EP

Kind code of ref document: A1