WO2000001843A1 - Analyse de sequence d'une matiere saccharide - Google Patents

Analyse de sequence d'une matiere saccharide Download PDF

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WO2000001843A1
WO2000001843A1 PCT/GB1999/002137 GB9902137W WO0001843A1 WO 2000001843 A1 WO2000001843 A1 WO 2000001843A1 GB 9902137 W GB9902137 W GB 9902137W WO 0001843 A1 WO0001843 A1 WO 0001843A1
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saccharide
chains
chain fragments
fragments
idoa
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PCT/GB1999/002137
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English (en)
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John Thomas Gallagher
Malcolm Lyon
Catherine Louise Ruby Merry
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Cancer Research Campaign Technology Limited
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Priority to AU46354/99A priority Critical patent/AU4635499A/en
Publication of WO2000001843A1 publication Critical patent/WO2000001843A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention is concerned with sequence analysis of saccharide material and it is especially applicable to the sequencing of saccharide chains composed of alternate glucosamine and hexuronate residues such as, for example, are found in glycosaminoglycans (GAGs) which include the biologically important polysaccharides heparan sulphate (HS) and heparin.
  • GAGs glycosaminoglycans
  • HS heparan sulphate
  • heparin heparin
  • Heparan sulphate (HS) and heparin are chemically-related linear glycosaminoglycans (GAGs) composed of alternate ⁇ , ⁇ -linked glucosamine and hexuronate residues with considerable structural variation arising from substitution with acetyl and N- and O-sulphate groups, and from the presence of D- and L-isomers of the hexuronate moieties.
  • GAGs linear glycosaminoglycans
  • These polysaccharides are of fundamental importance for many diverse cellular and biochemical activities. Their regulatory properties are dependent on their ability to bind, and in some cases cases to activate, protein molecules which control cell growth, cell adhesion, and enzyme-mediated processes such as haemostasis and lipid metabolism.
  • analysis of protein-binding monosaccharide sequences in HS/heparin is generally difficult and a universal procedure suitable for routine use has not been described to date.
  • An object of the present invention is to provide a new method of sequence analysis of saccharide fragments such as oligosaccharides that may be derived from HS (or heparan sulphate proteoglycan HSPG) and heparin, this method enabling rapid elucidation of recognition sites and other sequences of interest and thereby facilitating the rational design of synthetic compounds to serve as drugs for therapeutic modulation of polysaccharide function.
  • saccharide fragments such as oligosaccharides that may be derived from HS (or heparan sulphate proteoglycan HSPG) and heparin
  • exoenzymes in particular exoglycosidases
  • exoglycosidases for removal of terminal sugar residues at the non-reducing end of saccharide chains
  • methods for sequencing such chains described in other prior art documents, for instance in WO 92/02816 and in WO 92/19974 and WO 92/19768.
  • WO 92/02816 it was proposed in relation to a saccharide sequencing method disclosed therein to use exoenzymes successively to remove and identify terminal sugar residues at the non-reducing end of initially undegraded saccharide chains, and to carry out a series of sequential steps with the residual saccharide material being recovered after each step before proceeding to the next.
  • exoenzymes are mentioned inter alia as possible sequencing agents, again it was proposed that these be applied sequentially direct to an oligosaccharide being analysed in an iterative process without a preliminary partial depolymerisation step as required by the present invention.
  • this sequence can be determined, at least in most cases, by using a method similar to that described in our aforesaid international patent application except that it involves labelling each of at least the disaccharide units of the saccharide chains or chain fragments without the requirement of providing only the saccharide units at the reducing end of the chains with a label or referencing feature. This is in order that substantially all of the fragments produced by the partial depolymerisation step are labelled.
  • the labelling can optionally be deferred until after the partial depolymerisation step.
  • the labelling which is performed after the partial depolymerisation step is either labelling substantially throughout the oligosaccharide chain of substantially every fragment, e.g. on substantially every monosaccharide or disaccharide unit or residue, or labelling only on the reducing end of substantially all of the fragments.
  • substantially all of fragments produced by the partial depolymerisation step are labelled because it is not possible to guarantee that all fragments are labelled. This does not matter as long as a sufficient number of each type of fragment is labelled so that they each can subsequently be detected.
  • the method of the present invention involves a stage of partial depolymerisation of substantially homogeneous saccharide material composed of saccharide chains of the same molecular size and composition, this partial depolymerisation being carried out by controlled treatment of the saccharide material, suitably purified and/or fractionated, with a selective scission reagent that acts in accordance with a known predetermined linkage specificity to cleave a proportion of susceptible internal glycosidic linkages, that is, susceptible glycosidic linkages spaced from the non-reducing end of the saccharide chains and located along the length of the latter, thereby to produce a mixed set of saccharide chains, intact chains and fragments of intact chains, having different lengths representative of the full spectrum of all possible lengths given the particular glycosidic linkage specificity of the selective scission reagent employed.
  • samples of the mixed set of saccharide chains and chain fragments produced are treated, either singly or in combination, with selected exoenzymes comprising exosulphatases and exoglycosidases of known specificity that cleave only particular glycosidic linkages or particular sulphate groups at the non-reducing end of saccharide chains to an extent sufficient to obtain substantially complete digestion and cleave susceptible linkages at the non-reducing end of all the saccharide chains in each of said samples.
  • the samples treated with the exoenzymes, together with a control or reference sample of the partially depolymerised material not treated with said exoenzymes, are subjected to an analytical procedure whereby all of the labelled chains and chain fragments generated by the partial depolymerisation and enzymatic cleavage are resolved or separated spatially to produce a detectable signal pattern which is representative of the size and composition of the chain fragments and which provides an indication of any removal or modification of saccharide residues at the non-reducing end of the chains or chain fragments after the aforesaid exoenzyme treatment whereby substantially the entire monosaccharide sequence of the material, or at least the major part thereof, can be deduced without need to rely on a single end referencing feature per chain as used in WO 96/13606.
  • the signal pattern produced is a visual signal pattern and information for deducing the sequence of the saccharide units is obtained by visually comparing or reading the changes in the pattern produced by the different exoenzymes or combinations of exoenzymes. In so reading the pattern one will generally first consider changes in the pattern produced by the largest fragments generated in the partial depolymerisation stage, followed by then considering changes in the pattern produced by the next largest size of fragments and continuing progressively to do likewise for the next remaining fragments or sets of fragments until reaching the smallest size fragments.
  • the method of this invention is especially applicable to analysing and sequencing saccharide material comprising saccharide chains which contain more than three monosaccharide units interconnected through glycosidic linkages that are not all identical.
  • the method can be used reliably for sequencing chains containing up to at least twelve sugars or monosaccharide residues and, in some cases, even larger chains.
  • the saccharide material will generally be treated, usually before the controlled partial depolymerisation step, to modify the saccharide chains in order to introduce the required labelling feature in respect of substantially all the disaccharide units for facilitating, during analysis, detection of chain fragments derived from the original saccharide material.
  • the labelling operation could alternatively be carried out, if so desired, after the partial depolymerisation stage although usually such post-labelling of the fragments will not be so convenient.
  • This labelling feature can be provided by labelling or tagging the reducing end of the fragments or one or both monosaccharide residues in each disaccharide unit using for example radiochemical.
  • a radio labelled saccharide unit or precursor such as ⁇ -glucosamine and/or 35 S- sulphate may be added to the culture medium to provide metabolic radiolabelling.
  • electrophoretic separation means such as polyacrylamide gel electrophoresis (PAGE), e.g. gradient PAGE, will usually be used for detecting the fragments produced by the cleavage treatments, these fragments being separated to form a distinctive signal pattern according to differences in length and composition which are reflected in different mobilities in the electrophoretic medium.
  • PAGE polyacrylamide gel electrophoresis
  • the material can be treated in a preliminary operation so as to incorporate therein suitable electrically charged groups in a known manner in order to permit the use of electrophoretic separation techniques.
  • HPLC high performance liquid chromatography
  • the mixed set of saccharide chain fragments produced will usually be used to provide a number of separate samples.
  • one or more samples of the partially depolymerised material not subjected to exoenzyme treatment, and generally a control sample of the original material will be subjected to the separation technique, e.g. gradient PAGE or HPLC, to separate and detect the different fragments present for reference purposes before exoenzyme treatment.
  • the separation technique e.g. gradient PAGE or HPLC
  • other samples of the set of fragments will also be subjected to the same separation technique so as to separate and detect the different saccharide fragments present after each of these other samples has been treated with a different exoenzyme or combination of exoenzymes.
  • the preliminary controlled partial depolymerisation involving cleavage of specific internal glycosidic linkages is most conveniently carried out as hereinafter more fully described using nitrous acid at low pH as a chemical selective scission reagent. It is also possible, however, in some cases as an alternative to a chemical selective scission agent to use appropriate enzymatic endoglycosidases, e.g. the bacterial lyases heparinase (EC 4.2.2.7) or heparitinase (EC 4.2.2.8), under suitable conditions to bring about selective enzymatic cleavage of internal glycosidic linkages.
  • appropriate enzymatic endoglycosidases e.g. the bacterial lyases heparinase (EC 4.2.2.7) or heparitinase (EC 4.2.2.8
  • the selected exoenzymes used for treating the fragments obtained after the initial hydrolysis and partial depolymerisation will usually include, in addition to exoglycosidases, selected exosulphatases for effecting a controlled removal of particular sulphated groups from specific terminal monosaccharide residues at the non-reducing end of the chains or chain fragments.
  • Other additional specific enzymes may also be used in analysing the fragments obtained after the partial depolymerisation as part of the overall strategy selected for extracting or confirming the sequence information required.
  • the enzymes mentioned above are exoenzymes which act specifically to remove the terminal sugar residues or their sulphate substituents at the non-reducing end (NRE) of glycan fragments. Details of many such enzymes are readily available in the literature, and by way of example reference may be had to an informative review article entitled “Enzymes that degrade heparin and heparan sulphate” by John J. Hopwood in “Heparin: Chemical and Biological Properties, Clinical Applications", pages 191 to 227, edited by D.A. Lane et al.
  • Nitrous acid (HNO 2 ) reagent used at low pH cleaves hexosaminidic linkages when the amino sugar is N-sulphated (Glc ⁇ SO,) irrespective of the position of the linkage in the saccharide chain, but most importantly Glc ⁇ Ac — > GlcA linkages are resistant to H ⁇ O 2 scission.
  • Controlled hydrolysis and partial depolymerisation with nitrous acid can be achieved by preparing the reagent as described by Steven Radoff and Isidore Danishefsky, J. Biol. Chem. (1984), 259, pages 166-172, a publication of which the content is also incorporated herein by reference. A typical example with practical details, however, is described below.
  • the saccharide to be treated (1-2 nmoles) is dried down by centrifugal evaporation, dissolved in 80 L of distilled H 2 O and cooled on ice. To this solution is added 10 L of 190mM HCI and 10 L of lOmM NaNO 2 , both precooled on ice. These reactants are mixed by vortexing and incubated on ice. At predetermined time points (for example 0, 20, 40, 60, 90 and 120 minutes), aliquots of the reaction mixture are removed and the low pH HNO 2 hydrolysis is stopped, either by addition of excess ammonium sulphamate to quench the reagent or by raising the pH above 4.0 (for example by addition of Na 2 CO 3 solution).
  • HS heparan sulphate
  • FIGURE 2 is a chart or diagram illustrating the visual signal pattern that may be expected by electrophoretic separation and analysis using PAGE in relation to the set of saccharide chain fragments derived from the decasaccharide structure shown in FIGURE 1 following partial nitrous acid hydrolysis and exoenzyme treatment in accordance with the invention;
  • FIGURE 3 shows the sequence of a hexasaccharide that is the subject of a second example in which the individual sugar residues are labelled a to f;
  • FIGURE 4 (panels A to D) shows diagrams showing plots representing the patterns obtained in subjecting the hexasaccharide of FIGURE 3 to HPLC after partial depolymerisation by nitrous acid and exoenzyme treatment in accordance with the present invention;
  • FIGURE 5 shows preparative SAX HPLC chromatography of sized HS oligosaccharides. Sized dp6, d ⁇ 8 or dp 10 oligosaccharide populations were applied to a single ProPac PA-1 column, eluted with a linear gradient of NaCl in MilliQ water, pH 3.5 at a flow rate of lml/min, and 0.5ml fractions were collected. The gradients used were: dp6 (A), 0-1M NaCl over 80min; dp8 (B), 0-1.2M NaCl over 90min; dplO (C), 0-1.2M NaCl over 180min. For dp 10 only, no fractions were collected for the first 46 minutes. Elution profiles were monitored by scintillation counting ( 3 H, solid line, 3:, S, dashed line), and individual peaks pooled sharply (one or two central fractions only).
  • FIGURE 6 shows sequence analysis of hexasaccharide 6a.
  • A fragment profile generated by partial nitrous acid scission resolved on a ProPac PA-1 column eluted with a linear 0-0.75M NaCl gradient over 1 lOmins. Aliquots of these fragments (>5 Kcpm of 3 H per run) were subsequently digested with iduronate-2-sulphatase (B), iduronidase (C), or both enzymes sequentially (D). Fractions (0.5 min) were collected and counted for radioactivity ( H, solid line; 35 S, dashed line).
  • peaks are: nonsulphated disaccharide pool (nonS, fraction 18), free 35 SO 4 (fraction 38), free GlcNAc (NAc, fraction 5), disaccharide IdoA(2S)-aMan (fraction 45), tetrasaccharide R4, tetrasaccharide U4 and intact hexasaccharide 6a (fraction 1 18).
  • FIGURE 7 shows sequence analysis of octasaccharide 8a.
  • A fragment profile generated by partial nitrous acid resolved on a ProPac PA-1 column eluted with a linear 0-0.75M NaCl gradient over 1 10 mins. Aliquots of these fragments (>5Kcpm of 3 H per run) were subsequently digested with iduronate-2-sulphatase (B), iduronidase (C), or both enzymes sequentially (D). Fractions (0.5min) were collected and counted for radioactivity ( ⁇ , solid line).
  • peaks are: nonsulphated disaccharide pool (fraction 18), free 35 SO 4 (fraction 38), disaccharide IdoA(2S)-aMan (fraction 45), tetrasaccharide R4, tetrasaccharide U4, tetrasaccharide M4, hexasaccharide R6, hexasaccharide U6 and intact octasaccharide 8a (fraction 165).
  • FIGURE 8 shows sequence analysis of decasaccharide 10a.
  • A fragment profile generated by partial nitrous acid resolved on a ProPac PA-1 column eluted with a linear 0-0.75M NaCl gradient over HOmins. Aliquots of these fragments (>5 Kcpm of H per run) were subsequently digested with iduronate-2-sulphatase (B), iduronidase (C), or both enzymes sequentially (D). Fractions (0.5 min) were collected and counted for radioactivity ( 3 H, solid line; 35 S, dashed line).
  • peaks are: nonsulphated disaccharide pool (fraction 18), free " SO 4 (fraction 38), disaccharide IdoA(2S)-aMan (fraction 45), tetrasaccharide R4, tetrasaccharide U4, tetrasaccharide M4, hexasaccharide R6, hexasaccharide U6 and intact decasaccharide 10a (fraction 198).
  • FIGURE 9 shows sequence analysis of octasaccharide 8d.
  • A fragment profile generated by partial nitrous acid resolved on a ProPac A-l column eluted with a linear
  • peaks are: nonsulphated disaccharide pool (fraction 18), free 35 S0 4 (fraction 38), disaccharide IdoA(2S)-aMan (fraction 45), disaccharide IdoA(2S)-aMan(6S) (ISMS, fraction 118) tetrasaccharide R4, hexasaccharide U6, hexasaccharide R6.
  • the sequencing method of the present invention will naturally be applied to saccharide material composed of oligosaccharide chains having an unknown sequence of sugar residues, and since the oligosaccharide chains to be sequenced by the sequencing method of this invention need to be initially substantially homogeneous with respect to size and composition it will generally be necessary to purify the saccharide material to near homogeneity in a preliminary operation before sequencing.
  • This purification may be carried out, before or after labelling, using methods such as dialysis, weak anion exchange chromatography or strong anion exchange HPLC, gel filtration chromatography fractionation and/or, after labelling, gradient PAGE.
  • the bands can be electro-transferred from the gel to a positively charged nylon membrane and then dissociated from the membrane by incubation in 5M sodium chloride solution in a microcentrifuge tube on a rotating mixer at 37°C for 5 hours, and can then be desalted by chromatography of the solution on for example, HiTrapTM Desalting columns.
  • This approach using gradient PAGE resolves many saccharides more effectively than other methods and it is often a method of choice for preparing homogenous saccharide species for sequencing. Saccharides derived from HS produced by cultured cells can often be purified to a satisfactory level of homogeneity by gel filtration on a Bio Gel P-10 column followed by further separation using strong anion exchange chromatograph (Figure 3).
  • the sequencing method of the present is considered to be applied to the hypothetical decasaccharide structure shown in FIGURE 1 wherein all the disaccharide units are N-sulphated (except the one at the reducing end) and susceptible to cleavage by nitrous acid.
  • the saccharide material is first treated with nitrous acid under conditions that bring about partial scission of the hexosaminidic linkages as hereinbefore described, controlled so as to produce a mixed set of saccharide chains and fragments thereof having different lengths and sugar composition representative of the full spectrum of all possible lengths and compositions derivable from this saccharide structure given the particular linkage specificity of the nitrous acid selective scission reagent.
  • the nitrous acid partial depolymerisation will generate two different octamers, sugar residues A-H and C-J, three different hexamers A-F, C-H and E-J, four different tetramers A-D, C-F, E-H and G-J, and five dimers (disaccharides) A-B, C-D, E-F, G-H and I-J of which two, A-B and G-H, are identical in this particular example.
  • samples preferably aliquots, of the nitrous acid treated partially depolymerised saccharide material are then incubated separately with the following exoenzymes or combinations of exoenzymes: (a) glucuronidase (Gase) - acts on GlcA
  • I2Sase iduronate-2-sulphatase
  • each of the exoenzyme digests and the non- enzyme treated nitrous acid hydrolysate, and also the original saccharide material are analysed separately by polyacrylamide gel electrophoresis or high pressure anion exchange chromatography, i.e. PAGE or HPLC.
  • the theoretical banding patterns in different tracks after PAGE of the labelled fragments produced by these chemical and enzymic treatments in this example of the structure of FIGURE 1 may be substantially as shown in FIGURE 2. Based on the radiolabelling feature this is presented as a visual signal pattern, using for example electrotransfer to nylon membrane material and fluorography.
  • pHNO 2 + iduronidase (Idase) pHNO 2 + iduronate-2-sulphatase (I2Sase)
  • Running conditions for such gel electrophoresis may be as described previously in the literature, e.g. Turnbull and Gallagher (1988) Biochem J. 251, 597- 608.
  • the migration banding pattern depicted in Figure 2 reflects the different mobilities of saccharides with 4, 6, 8 and 10 sugar units (dp4-10). The actual mobilities of each saccharide can be determined experimentally.
  • Disaccharides would of course also be produced and separated, but in practice they may not be readily visible and visualisation may not be necessary given additional data from chemical analysis. For simplicity they are therefore not shown in FIGURE 2.
  • the slowest moving band (at top) in track (1) represents the original saccharide sample. This is the sample A-J, corresponding to dp 10.
  • Track (1) also shows that all the amino sugars in this saccharide are N- sulphated, i.e. two octasaccharides. three hexasaccharides etc. (this would be confirmed by the disaccharide analysis).
  • N- sulphated i.e. two octasaccharides. three hexasaccharides etc. (this would be confirmed by the disaccharide analysis).
  • GlcNSO residue at positions B, D, F and H.
  • Track (2) shows GlcA at position A - (the original 10 mer moves; but since only one 8 mer moves, position C does not contain GlcA).
  • Track (3) shows IdoA at position E - (as there is no shift of 8 mers, the shift of one 6 mer places IdoA at position E).
  • Track (4) shows an iduronate-linked 2-sulphate at position C (n.b. only one 8 mer moves, the original 10 mer, as expected, fails to move).
  • HPLC resolution provides signals in the form of peaks, representative of the different species of saccharide fragments, that are plotted graphically as shown in the panels A to D of FIGURE 4.
  • panel 4A shows the plot obtained for the partial nitrous acid (pHNO 2 ) hydrolysis only.
  • the labels on the peaks in the diagram signify the following:
  • the two tetramers produced correspond to fragments a - d and c - f (see
  • FIGURE 3 The a - d structure is U4, the so-called non-mover because it cannot be degraded by the exo-enzymes due to its unsaturated uronate at the non-reducing end. Thus, it will be seen that the U4 peak appears in the same position in all plots.
  • Structure c- f corresponds to peak E4, the mover; this moves with treatment by iduronate-2-sulphatase (panel B; 2S-ase), but not with treatment by iduronidase (Ido- ase) alone (panel C). It does move appreciably, however, with the combination of Ido-ase/2S-ase shown in panel D (peak designation E4").
  • the structure c - f corresponding to E4 has IdoA,2S at the end corresponding to residue c.
  • iduronidase splits the disaccharide peak (panel C) to yield an early-eluting peak at about fraction 5.
  • oligosaccharides were partially depolymerised using dilute nitrous acid. Each oligosaccharide was subjected to partial depolymerisation using dilute nitrous acid as described (Radoff, S., and Danishefsky, I. (1984) J. Biol. Chem. 259, 166-172), with aliquots of the reaction being stopped at a number of time points to generate a range of intermediates of the depolymerisation process. Briefly, each oligosaccharide was lyophilised, then resuspended in 80 ⁇ l of H 2 O to which was added 10 ⁇ l each of lOmM NaNO 2 and 190 mM HCl.
  • samples to be digested with enzymes were desalted by passage over PD-10 size exclusion columns eluted with H 2 _ O, then lyophilised.
  • Digests were set up in a total volume of 25 ⁇ l of 40 mM sodium acetate, pH 4.5. Iduronate-2-sulphatase and iduronidase were used either singly, or combined sequentially. Each individual enzyme digest was incubated for 12 h at 37°C. Iduronidase was used at 0.29 mU per digest and iduronate-2-sulphatase was used at 0.54 mU per digest. After treatment with the enzymes, samples were adjusted to 1ml by addition of H 2 O/HCl pH 3.5.
  • FIG. 6A A typical profile is shown for the major hexasaccharide species designated 6a ( Figure 6A).
  • peaks which correspond to the nonsulphated disaccharides (fraction 18, labelled non-S; IdoA-aMan. GlcA-aMan and ⁇ UA-aMan all co-elute as a single peak), the sulphated disaccharide IdoA(2S)-aMan (fraction 45, labelled ISM), free ,5 SO 4 (fraction 38).
  • two tetrasaccharides fraction 78 and 95, designated R4 and U4 in Table 1 and the original hexasaccharide at fraction 118.
  • the disaccharides were identified by comparison with known standards. R4 and U4 were confirmed as tetrasaccharides by their size elution position on a Bio-Gel P-10 column (data not shown). The appearance of two tetrasaccharides indicates that there are two internal GlcNS residues in dp6a.
  • One of these tetrasaccharide fragments, in common with the original dp6 fragment, will contain a ⁇ 4,5 unsaturated uronate at its non-reducing end and will be resistant to lysosomal enzymes, the other will be linked to the reducing end and will be susceptible to the enzymes.
  • the disaccharide identified as IdoA(2S)-aMan moves as expected after iduronate-2-sulphatase treatment, but not with iduronidase alone, providing a useful internal control for the action of enzymes.
  • the last piece of information to be extracted from the profiles is the identity of the residues in the reducing terminal disaccharide of dp ⁇ a.
  • ⁇ UA-GlcNAc was seen in the disaccharide analysis of the hexsaccharide, and iduronidase generates a peak in the position of free GlcNAc from the non-sulphated disaccharides. Nitrous acid scission showed that all the internal amino sugars were N-sulphated, therefore the deduced IdoA-GlcNAc disaccharide must be at the reducing end.
  • the entire sequence of dp ⁇ a is therefore:
  • N-acetylated disaccharide is also present in dp8a, and this must therefore be at the reducing end. This is compatible with the fact that iduronidase yields free
  • dp8a The complete sequence of dp8a is therefore: ⁇ UA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-GlcNS-IdoA-GlcNAc.
  • panels B-D in Figure 9 represent the profiles for iduronate-2-sulphatase (B), iduronate-2-sulphatase and iduronidase (C), or both enzymes sequentially, followed by 6-sulphatase (D).
  • B iduronate-2-sulphatase
  • C iduronate-2-sulphatase
  • D 6-sulphatase
  • Fragment R6 therefore contains a non-reducing terminal sequence of IdoA(2S)-GlcNS(6S) and is a reducing end fragment of dp8d.
  • R4 in 6a, 8a, and 1 Oa helped to confirm the sequences.
  • the invention provides a number of different aspects and, in general, it embraces all novel and inventive features and aspects herein disclosed either explicitly or implicitly and either singly or in combination with one another. Moreover, the scope of the invention is not to be construed as being limited by the illustrative examples or by the terms and expressions used herein merely in a descriptive or explanatory sense, and many modifications may be made within the scope of the invention defined in the appended claims.
  • the invention has been described mainly in relation to saccharides that are found in heparan sulphate and heparin, the basic principle of the sequencing strategy is applicable to many other GAGs and different saccharides, including the saccharide component of glycoproteins. It will also be appreciated that with substantially all of the saccharide chains and chain fragments being labelled in carrying out the method of this invention, with the appropriate choice of exoenzymes sequencing could also be carried out in the opposite direction, i.e. from the reducing end towards the non-reducing end. Thus, the invention can provide a bidirectional sequencing method.

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Abstract

La présente invention concerne une technique d'analyse et de séquençage appliquée à une matière saccharide renfermant des chaînes sacharride à plus de trois unités monosacharrides dans le but d'obtenir des informations sur les séquences. Cette technique consiste à: (a) soumettre ladite matière saccharide à une dé-polymérisation partielle à traitement contrôlé au moyen d'un réactif de scission sélective pour obtenir un ensemble mixte de chaînes saccharide, de chaînes intactes et de fragments de chaînes intactes dans des longueurs différentes représentatives de toute la plage des longueurs possibles; (b) traiter un échantillon dudit ensemble mixte de chaînes saccharide et de fragments de chaîne suffisamment avec lesdites exoenzymes pour obtenir une digestion complète et des liaisons susceptibles de clivage à l'extrémité non réductrice de toutes les chaînes saccharide et des fragments de chaînes;(c) analyser le ou les échantillons en question afin de détecter les divers fragments de chaîne saccharide engendrés par les opérations de clivage qui sont présents dans le ou les échantillons et de recueillir, collectivement à partir des résultats de l'analyse, des informations permettant de déduire au moins en partie la séquence monosaccharide de la matière saccharide d'origine. Ce processus s'accompagne pour l'essentiel du marquage de toutes les chaînes saccharide et tous les fragments de chaîne.
PCT/GB1999/002137 1998-07-03 1999-07-05 Analyse de sequence d'une matiere saccharide WO2000001843A1 (fr)

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US8409604B2 (en) 2006-05-12 2013-04-02 W. L. Gore & Associates, Inc. Immobilized biologically active entities having a high degree of biological activity
US8496953B2 (en) 2006-05-12 2013-07-30 W. L. Gore & Associates, Inc. Immobilized biologically active entities having a high degree of biological activity following sterilization
US8691260B2 (en) 2006-05-12 2014-04-08 W. L. Gore & Associates, Inc. Immobilized biologically active entities having a high degree of biological activity
US8945599B2 (en) 2006-05-12 2015-02-03 W. L. Gore & Associates, Inc. Immobilized biologically active entities having a high degree of biological activity
US8986713B2 (en) 2006-05-12 2015-03-24 W. L. Gore & Associates, Inc. Medical device capable of being compacted and expanded having anti-thrombin III binding activity
US9114194B2 (en) 2006-05-12 2015-08-25 W. L. Gore & Associates, Inc. Immobilized biologically active entities having high biological activity following mechanical manipulation
US9375515B2 (en) 2006-05-12 2016-06-28 W. L. Gore & Associates, Inc. Immobilized biologically active entities having high biological activity following mechanical manipulation
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WO2011035001A3 (fr) * 2009-09-17 2011-08-18 Gore Enterprise Holding, Inc. Nouvelles entités à base d'héparine et méthodes d'utilisation de celles-ci
US9101696B2 (en) 2011-03-11 2015-08-11 W.L. Gore & Associates, Inc. Immobilised biological entities
US9408950B2 (en) 2011-03-11 2016-08-09 W.L. Gore & Associates, Inc. Immobilised biological entities
US9764068B2 (en) 2011-03-11 2017-09-19 W.L. Gore And Associates Inc. Immobilised biological entities
US10736999B2 (en) 2011-03-11 2020-08-11 W.L Gore & Associates, Inc. Immobilised biological entities
US11497838B2 (en) 2011-03-11 2022-11-15 W. L. Gore & Associates, Inc. Immobilised biological entities
CN111275644A (zh) * 2020-01-20 2020-06-12 浙江大学 一种基于Retinex算法的水下图像增强方法和装置

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