WO2000001843A1 - Sequence analysis of saccharide material - Google Patents

Abstract

The invention provides a method of analysing and sequencing saccharide material comprising saccharide chains which contain more than three monosaccharide units to obtain sequence information. The method comprises the sequential steps of: (a) subjecting said saccharide material to partial depolymerisation by controlled treatment with a selective scission reagent 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; (b) treating a sample of said mixed set of saccharide chains and chain fragments with said exoenzymes to an extent sufficient to obtain complete digestion and cleave susceptible linkages at the non-reducing end of all the saccharide chains and chain fragments and chain fragments and then (c) analysing said sample or samples to detect the various saccharide chain fragments generated by the cleavage treatments which are present therein, and obtaining, collectively from the results of said analysis, information enabling the monosaccharide sequence in the original saccharide material to be at least partially deduced; wherein substantially all of the saccharide chains and chain fragments produced by step (a) are labelled.

Description

SEQUENCE ANALYSIS OF SACCHARIDE MATERIAL

FIELD OF THE INVENTION

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.

BACKGROUND Heparan sulphate (HS) and heparin are chemically-related linear glycosaminoglycans (GAGs) composed of alternate α,β-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. 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. However, 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.

SUMMARY OF THE INVENTION

In International Patent Application No. PCT/GB95/02541, Publication No. WO 96/13606, we have previously described a method of sequence analysis of saccharide material based on a concept of bringing about a preliminary partial depolymerisation by scission of specific intrachain linkages in reducing end referenced saccharide chains, such as for example HS/heparin saccharide chains, followed by exoenzyme removal of non-reducing end (NRE) sugars or their sulphate groups so as to yield a range of labelled fragments that can be separated by gel electrophoresis or other appropriate techniques to give a read-out of the sequence of sugar units and their substituents.

Use of exoenzymes, in particular exoglycosidases, for removal of terminal sugar residues at the non-reducing end of saccharide chains has also been proposed previously in connection with 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. However, in these other prior art proposals there has been no preliminary step of partial depolymerisation of the saccharide material, involving cleavage of internal glycosidic linkages, before treatment with said exoenzymes. In WO 92/02816 for example, 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. In WO 92/19974 and WO 92/19768, although 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. There is also a paper by Kyung-Bok Lee et al., (1991), Carbohydrate Research, 214, 155-168, which refers to the use of exoglycosidases and of endoglycosidases in connection with sequencing of oligosaccharides. This publication does not, however, disclose the combined use of both exoglycosidases and endoglycosidases in sequence in the same manner as herein described in relation to the present invention.

A feature of the method described in our aforesaid international patent application, at least insofar as it applied to sequence analysis of oligosaccharide chains composed of alternate but variable glucosamine and hexuronate residues in unknown sequence, was the fact that the saccharide chains were labelled or end referenced at, but only at, their reducing end. It has now been found, however, that even although there may be different hexuronate residues along the length of the saccharide chains so that there is variation in disaccharide composition within an initially unknown sequence of monosaccharide residues, 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. Thus, as an alternative 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.

It is stated that 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.

As in the method of our aforesaid international patent application 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.

After the partial depolymerisation stage 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. In the next stage 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.

Preferably 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. In general there will be a plurality of disaccharide units composed of linked glucosamine and hexuronate residues with structural variation along the length of the chains. 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.

In carrying out this saccharide sequencing method of the present invention, 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. It should be noted, however, that 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. fluorescent, biotin or other colorimetrically detectable labelling means. When the saccharide material is derived from cells grown in culture, a radio labelled saccharide unit or precursor such as Η-glucosamine and/or ³⁵S- sulphate may be added to the culture medium to provide metabolic radiolabelling.

In some preferred embodiments, especially for longer oligosaccharide chains, as hereinafter more fully described, 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. If necessary, for uncharged or lightly charged saccharide chains, 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. This will not usually be necessary, however, in sequencing HS or heparin oligosaccharides which already contain a significant number of charged sulphate and carboxyl groups. In other preferred embodiments, however, alternative separation techniques, for example capillary electrophoresis or high performance liquid chromatography (HPLC), may be used for detecting the fragments and for providing a distinctive signal pattern, for example in the form of a pattern of peaks in an analysis output display or printout. In some cases, HPLC is the especially preferred analytical technique.

After the controlled partial depolymerisation step the mixed set of saccharide chain fragments produced will usually be used to provide a number of separate samples. In a typical procedure, 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. At the same time, 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.

In applying the invention to the sequencing of saccharide chains containing many amino sugar residues, such as are found in glycosaminoglycans (GAGs) for which the method is especially useful, 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.

As GAGs and similar saccharides also generally contain various sulphated monosaccharide units, 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.

Examples of selective scission reagents which may be used in carrying out the sequencing method of the present invention include the following:

Reaεents Linkage Specificity

(1) *Nitrous Acid GlcNSO₃ ► HexA

(2) *Glucuronidase (Gase) GlcA GlcN.R

(β-D-glucuronidase)

(3) *Iduronidase (Idase) IdoA •GlcN.R

(α-L-iduronidase)

(4) */v^r-acetylglucosaminidase GlcNAc HexA

(5) ♦ Iduronate-2-Sulphatase (I2Sase) IdoA — ^•GlcN.R

2S

(6) ♦ Glucosamine-6-Sulphatase (G6Sase) GlcN.R iVHexA

6S

e-g- ♦ N-acetylglucosamine-6-sulphatase GlcΝAc ► IdoA

6S

(7) ♦ Glucuronate-2-Sulphatase GlcA GlcN.R

2S

(8) ♦ Sulphamate sulphohydrolase GlcΝSO, ► HexA

Abbreviations and labels used above have the following meanings:

GlcΝ. = Glucosamine

R = Acetyl (Ac) or SO HexA = Hexuronic acid

GlcA = Glucuronate

IdoA = Iduronate

* Cleaves glycosidic linkages

♦ Removes sulphate groups only

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. and published by Edward Arnold, London, 1989, and to another review article entitled "Lysosomal Degradation of Heparin and Heparan Sulphate" by Craig Freeman and John Hopwood in "Heparin and Related Polysaccharides", pages 121 to 134, also edited by D.A. Lane et al. and published by Plenum Press, New York, 1992.

Some of these enzymes are available commercially and others can be isolated and purified from natural sources as described in the literature. Moreover, in some cases recombinant versions are known and are available; these will often be preferred because of a high level of purity that can usually be achieved. Published papers in which the isolation and preparation or properties of some of the enzymes referred to above are described include: Alfred Linker, (1979), "Structure of Heparan Sulphate Oligosaccharides and their Degradation by Exo-enzymes", Biochem. J., 183, 71 1-720; Craig Freeman and John J Hopwood, (1992), "Human α-L-iduronidase", Biochem. J., 282, 899-908; Wolfgang Rohrborn and Kurt Von Figura, (1978), "Human Placenta α-N- Acetylglucosaminidase: Purification, Characterisation and Demonstration of Multiple Recognition Forms", Hoppe-Seyler's Z. Physiol. Chem., 359, 1353-1362; Craig Freeman and John J Hopwood, (1986), "Human Liver Sulphamate sulphohydrolase", Biochem. J, 234, 83-92; Craig Freeman, et al, (1987), "Human Liver N- acetylglucosamine-6-sulphate sulphatase", Biochem. J, 246, 347-354; Craig Freeman and John J Hopwood, (1991), "Glucuronate-2-sulphatase activity in cultured human skin fibroblast homogenates", Biochem. J., 279, 399-405; Craig Freeman and John J Hopwood, (1987), "Human liver N-acetylglucosamine-6-sulphate sulphatase", Biochem. J., 246, 355-365; Irwin G. Leder (1980), "A novel 3-0 sulfatase from human urine acting on methyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3- sulphate", Biochemical and Biophysical Research Communications, 94, 1 183-1189; Julie Bielicki, et al, (1990), "Human liver iduronate-2-sulphatase", Biochem. J., 271, 75-86; and Ulf Lindahl et al, (1980), "Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin", Biochemistry, 77, 6551- 6555.

A recombinant version of an exoenzyme and the preparation thereof is described for example in connection with a synthetic α-L-iduronidase in international patent publication WO 93/10244.

The contents of the above-mentioned publications are incorporated herein by reference.

Nitrous acid (HNO₂) reagent used at low pH cleaves hexosaminidic linkages when the amino sugar is N-sulphated (GlcΝSO,) irrespective of the position of the linkage in the saccharide chain, but most importantly GlcΝAc — > GlcA linkages are resistant to HΝO₂ 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.

Example of conditions for Nitrous Acid hydrolysis and partial depolymerisation of saccharides:

The saccharide to be treated (1-2 nmoles) is dried down by centrifugal evaporation, dissolved in 80 L of distilled H₂O and cooled on ice. To this solution is added 10 L of 190mM HCI and 10 L of lOmM NaNO₂, 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 HNO₂ 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 Na₂CO₃ solution). It has in fact been found most convenient to stop the reaction by addition of 1/4 volume of 200mM sodium acetate buffer, pH 5.0. Finally, once all the time points are complete the aliquots are remixed and pooled. This is crucial since it creates a mixed set of saccharide products, hydrolysed partially and at random, which contain fragments corresponding to all possible cleavage positions, whereas a single time point would not create such a representative set. Thus, the fragments have different lengths ranging throughout the full spectrum of possible lengths for the particular glycosidic linkage specificity of the HNO₂ reagent, and ideally there should be a fairly even distribution of the different length fragments. The samples are then desalted, e.g. on a Sephadex G-25 column, dried, then taken up in enzyme buffer and the enzymes are added as described later.

In carrying out the invention, it will be appreciated that in effect the controlled, incomplete hydrolysis of N-sulphated disaccharides by the HΝO₂ treatment, i.e. the partial HNO₂ scission or depolymerisation (herein denoted as pHNO₂), is used to "open-up" the glycan structure of the saccharide material under analysis so as to expose a range of NRE sugars and sulphate groups to attack by specific exoglycosidases and exosulphatases. This dual approach of combining a preliminary controlled hydrolysis and partial depolymerisation involving cleavage of internal linkages with a progressive action of exoenzymes acting at the non-reducing end of the fragments produced follows the approach described in WO 96/13606.

MORE DETAILED DESCRIPTION

The invention and the manner in which it may be carried out will now be hereinafter described in more detail with reference to non-limiting illustrative examples. Brief Description of the Drawings

In connection with the above-mentioned illustrative examples, reference should be made to the accompanying drawings in which:

FIGURE 1 represents a hypothetical but possible structure of a decasaccharide (degree of polymerisation dp=10) fragment that may be derived from heparan sulphate (HS) or heparin, wherein the individual monosaccharide residues at positions 1 to 10 are labelled A to J;

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, dρ8 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 (³H, 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 ³H 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; ³⁵S, dashed line). Identified peaks are: nonsulphated disaccharide pool (nonS, fraction 18), free ³⁵SO₄ (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 ³H 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 (Η, solid line). Identified peaks are: nonsulphated disaccharide pool (fraction 18), free ³⁵SO₄ (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 (³H, solid line; ³⁵S, dashed line). Identified peaks are: nonsulphated disaccharide pool (fraction 18), free ^" SO₄ (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

0-0.75M NaCl gradient over 110 mins. Aliquots of these fragments (>5 Kcpm of ³H per run) were subsequentially digested with iduronate-2-sulphatase (B), iduronate-2- sulphatase and iduronidase (C), or both enzymes sequentially, followed by 6- sulphatase (D). Fractions (0.5min) were collected and counted for radioactivity (³H, solid line; ³⁵S, dashed line). Identified peaks are: nonsulphated disaccharide pool (fraction 18), free ³⁵S0₄ (fraction 38), disaccharide IdoA(2S)-aMan (fraction 45), disaccharide IdoA(2S)-aMan(6S) (ISMS, fraction 118) tetrasaccharide R4, hexasaccharide U6, hexasaccharide R6.

In practice 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 latter technique can be particularly useful for purification purposes since it allows excellent resolution of labelled saccharides which can be readily recovered by electrotransfer to positively charged nylon membrane as described for example by Turnbull and Gallagher in Biochemical Journal (1988) 251, 597-608 and, with additional modifications, in Biochemical Journal (1991) 265, 715- 724. Accordingly, the content of these publications is incorporated herein by reference. In this technique, the appropriate bands in the electrophoresis gel are cut out, their position being established with reference to the label used. Alternatively, 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, HiTrap™ 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).

In the following two examples which are presented primarily to explain more clearly the principle of the sequencing method of the present invention by reference to hypothetical oligosaccharide structures, a decamer and a hexamer, such as may be derived by nitrous acid hydrolysis from sulphated glycosaminoglycans like heparan sulphate (HS) or heparin. It will be assumed for simplicity that the above-mentioned preliminary purification and labelling operations will have already been carried out, the latter being effected for example by metabolic radiolabelling using ³H- glucosamine and/or ^3:,S-sulphate if it be further assumed for example that the saccharide material is obtained from cells or tissues grown in culture.

EXAMPLE 1

In this example 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. To sequence this structure, experimentally 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. Thus, in this case it will be seen that 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.

In this 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

(b) iduronidase (Idase) - acts on IdoA

(c) iduronate-2-sulphatase (I2Sase) - acts on IdoA, 2S

(d) a combination of (b) and (c)

These enzymes, being exoenzymes, only act on sugars at the non-reducing end of the saccharide.

After this enzymic treatment 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. The basic method using polyacrylamide gel electrophoresis for separating the treated saccharides and producing a visual signal pattern showing the location of the saccharide chains or fragments in the gel (oligosaccharide mapping) is described in more detail by Turnbull and Gallagher in Biochemical Journal (1988), 251. 597-608 and Biochemical Journal (1991) 265, 715-724. In the chart or diagram of Figure 2 tracks 2-5 represent the enzyme treated samples and track 1 represents the separation of the complete set of pHNO₂ fragments without enzyme treatment. This read-out then allows at least most of the sequence to be read. To summarise, in Figure 2 the identity of the samples in the different tracks 1 to 5 is as follows:

(1) Partial HNO₂ hydrolysate of decasaccharide (pHNO₂)

(2) pHNO₂ + glucuronidase (G'ase)

(3) pHNO₂ + iduronidase (Idase) (4) pHNO₂ + iduronate-2-sulphatase (I2Sase)

(5) pHNO₂ + I2Sase + Idase

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.

Deducing the sequence

Returning to FIGURE 2 and interpretation thereof:

1. The slowest moving band (at top) in track (1) represents the original saccharide sample. This is the sample A-J, corresponding to dp 10.

2. 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). Thus, there is a GlcNSO, residue at positions B, D, F and H. To read the sequence, one now concentrates on the bands of the largest fragments in each track before stepping down to the smaller ones.

3. 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).

4. 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).

5. 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). By this stage there is the following sequence information, bearing in mind that wherever the iduronate-2-sulphatase acts, there must be an IdoA linked to it (this is confirmed in track 5).

GlcA-GlcNSO₃-IdoA-GlcNSO₃-IdoA-GlcNSO₃ I

2S

A B C D E F

The residue at position G can then be deduced from inspection of the tetramers

(next step down in fragment size). The only enzyme that causes two tetramers to shift is glucuronidase; and since a shift in one tetramer would be predicted from the GlcA already identified at position A, the other must be at position G. The residues at position I and J after nitrous acid hydrolysis would be readily apparent from a direct analysis of the disaccharide composition of the sample. Because this sequencing technique identifies all disaccharides except the one at the reducing end, the latter can be determined by comparison with the results of a quantitative analysis of disaccharide composition. Thus, one can arrive at a complete sequence without single end-labelling per chain as used in WO 96/13606. EXAMPLE 2

This relates to the actual sequencing of a hexasaccharide isolated from HS using the enzyme heparinase III (heparitinase) which cleaves mainly at GlcNAc/GlcNSO - GlcA linkages. This always leaves an unsaturated (Δ) uronic acid at the non-reducing end. Chemical analysis of the hexasaccharide indicated that it was composed of three different disaccharides, as follows:

UA — GlcNS

2S

UA — GlcNAc

UA — GlcNSO, where UA = glucuronic acid or iduronic acid

In the disaccharide analysis for technical reasons it is not possible to identify the uronic acid at the non-reducing end of each disaccharide unit and it could be either glucuronic acid or iduronic acid. However, except for the residue at the non-reducing end of the complete hexamer, the remainder of the sequence was fully identified by the sequencing method of the present invention using HPLC to separate the oligosaccharide chain fragments and produce a signal pattern giving information from which the saccharide sequence could be deduced.

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. Directing attention first to panel 4A, this shows the plot obtained for the partial nitrous acid (pHNO₂) hydrolysis only. The labels on the peaks in the diagram signify the following:

(6) - original hexamer

U4 - non-moving tetramer

E4 - enzyme susceptible tetramer

ISM - iduronate-2-sulphate-anhydromannose Δ UA-M \ two disaccharides co-eluting

I-NAc-

The presence of two tetramers in the pHNO₂ shows that the GlcNS residues must be at positions (b) and (d). If the GlcNAc residue were to be at either of these positions the pHNO₂ treatment would yield only one tetramer.

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"). Thus, it can be deduced that the structure c - f corresponding to E4 has IdoA,2S at the end corresponding to residue c. It will also be seen that iduronidase splits the disaccharide peak (panel C) to yield an early-eluting peak at about fraction 5. This confirms that one of the disaccharides must have iduronate at the end. It cannot be either residue a (ΔUA) or residue c (IdoA,2S), so it must be residue e. Thus we have the entire hexamer sequence as shown in FIGURE 3. This strategy has already been extended to sequencing of other 8 mers and 10 mers.

EXAMPLE 3

S-domains generated by heparinase III digestion were separated by Bio-Gel

P10 gel filtration and SAX- HPLC to yield a range of oligosaccharide species differing in length, sugar sequence, and sulphation pattern. The HPLC separations for dp6-10 (dp=degree of polymerisation, or number of saccharide units, e.g. dp6=hexasaccharide), are shown in Figure 5.

Individually purified oligosaccharides, from Figure 5, 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 μl of H₂O to which was added 10 μl each of lOmM NaNO₂ and 190 mM HCl. At each stop point (usually 30 min, 1 hr, 2 hr, 3 hr and 4 hr), 20 μl of the reaction mixture was removed and added to a common vial containing 25 μl of 0.2 M sodium acetate, pH 5.0. All procedures and reagents were at 4°C.

Following nitrous acid depolymerisation, samples to be digested with enzymes were desalted by passage over PD-10 size exclusion columns eluted with H₂_ O, then lyophilised. Digests were set up in a total volume of 25 μl 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 H₂O/HCl pH 3.5.

The scission products separated by SAX-HPLC using a single ProPac PA-1 SAX column (4.6 x 250mm), and a linear gradient running from 0-0.75 M NaCl over 1 10 min. Samples were loaded using a 1ml sample injection loop. Following sample loading, the loop was washed onto the column with 1ml of H₂O before application of the gradient. The column was eluted at a flow rate of 1ml min^"1 collecting 0.5 ml fractions. Radioactivity in each fraction was measured by scintillation counting.

A typical profile is shown for the major hexasaccharide species designated 6a (Figure 6A). In this profile, we can recognise peaks which correspond to the nonsulphated disaccharides (fraction 18, labelled non-S; IdoA-aMan. GlcA-aMan and ΔUA-aMan all co-elute as a single peak), the sulphated disaccharide IdoA(2S)-aMan (fraction 45, labelled ISM), free ^,5SO₄ (fraction 38). two tetrasaccharides (fraction 78 and 95, designated R4 and U4 in Table 1) and the original hexasaccharide at fraction 118.

TABLE 1

a b c d e f

ΔUA- GlcNSC-3- IdoA(2S)- GlcNSθ3 -IdoA- GlcNAc dp6 ΔUA- GICNSO3- IdoA(2S)- a an U4 ldoA(2S)- GlcNS0 -ldoA-GlcNAc R4 IdoA- GICNSO3 -IdoA- GlcNAc R4' GIcNS0₃ -IdoA- GlcNAc R4" ΔUA- aMan

IdoA(2S)- aMan

IdoA- aMan aMan

IdoA- GlcNAc

IdoA- GlcNAc

GlcNAc

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 Δ4,5 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. It is possible to distinguish between tetrasaccharides R4 and U4 by comparing the profiles in panels B-D in Figure 6. The profiles in panels B-D were obtained by digesting aliquots of the fragments (having profile A) with iduronate-2-sulphatase (B), iduronidase (C), or both enzymes sequentially. The results are shown in Table 2. TABLE 2

a b c d e F

ΔUA- GlcNS0₃- IdoA(2S)- GICNSO3 -IdoA- GlcNAc 6a

ΔUA- GlcNSO-3- IdoA(2S aMan U4

IdoA(2S)- GICNSO3 -IdoA- GlcNAc R4

IdoA- GIcNS0₃ -IdoA- GlcNAc R4'

IdoA(2S)- GICNSO3 -IdoA- GlcNAc R4

GICNSO3 -IdoA- GlcNAc R4"

Digestion with iduronate-2-sulphatase causes the R4 peak (at fraction 78) to shift to fraction 45 (R4¹); further digestion with iduronidase causes an additional shift to fraction 31 (R4"). This tetrasaccharide is therefore unequivocally identified as being derived from the Reducing end of dpόa (thus designated R4), and it contains a terminal IdoA(2S). The other tetrasaccharide is not affected by the enzymes, and is derived from the Unsaturated non-reducing end of dpόa (thus designated U4). As expected, the original dpόa is also unchanged after exo-enzyme digestion.

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 dpόa. ΔUA-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 dpόa is therefore:

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA-GlcNAc

The disaccharide analysis of dpόa (Table 3) is compatible with this sequence.

TABLE 3

ΔUA-GlcNAc ΔUA-GlcNS ΔUA(2S)-GlcNS ΔUA(2S)-GlcNS(6S) 6a 1.17 1.00 1.09

8a 0.99 1.00 2.13

8d 0.97 1.00 1.14 0.88

10a 0.94 1.00 2.92

EXAMPLE 4

Major octasaccharide 8a generated by heparinase III digestion and separated by Bio-Gel P10 gel filtration and SAX-HPLC was subjected to a similar analysis to that of Example 3, generating the profiles seen in Figure 7. The profiles in panels B- D were obtained by digesting aliquots of the fragments (having profile A) with iduronate-2-sulphatase (B), iduronidase (C), or both enzymes sequentially (D). The results are shown in Table 4.

TABLE 4 a b c d e f g h

ΔUA- GlcNSC-3- IdoA(2S)- GICNSO3- IdoA(2S)- GICNSO3 - IdoA- GlcNAc 8a

ΔUA- GIcNS0₃- IdoA(2S)- aMan U4

IdoA(2S)- GICNSO3 - IdoA- GlcNAc R4

ΔUA- GICNSO3- IdoA(2S)- GICNSO3- IdoA(2S)- aMan U6

IdoA(2S)- GIcNS0₃- IdoA(2S)- GlcNS0₃ - IdoA- GlcNAc R6

IdoA(2S)- GICNSO3- IdoA(2S)- aMan M4

IdoA- GlcNSO-3- ldoA(2S)- GICNSO3- IdoA-GlcNAc R6' IdoA- GICNSO3- IdoA-GlcNAc R4'

IdoA(2S GICNSO3 -IdoA(2S)-GlcNS03- IdoA-GlcNAc R6 IdoA(2S)- GlcNS0₃- IdoA-GlcNAc R4

GlcNSO-3- IdoA(2S)- GICNSO3- IdoA-GlcNAc R6"

GICNSO3- IdoA-GlcNAc R4"

Again, by observing the ways in which each of the peaks seen in the profile of the initial partial nitrous acid scission (Figure 7, panel A) are affected by the sequencing enzymes, it is possible to assign fragments for sequencing.

A number of peaks were observed but the key ones for sequencing are the hexa- and tetrasaccharides R6 and R4. The enzyme digestions (Panels B-D) show that both R6 and R4 contain terminal IdoA(2S) units. Another tetrasaccharide derived from the Middle of dp8a (thus designated M4), was also present after partial nitrous acid treatment and this fragment also contained an IdoA(2S) terminal unit), whereas a third tetrasaccharide (U4) was enzyme resistant and therefore from the unsaturated end. The presence of three tetrasaccharides (R4, M4 and U4) after partial nitrous acid shows that the three internal GlcN residues of dp8a are N-sulphated.

An 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

GlcNAc from the non-sulphate disaccharide peak. Furthermore, the elution characteristics of R4 in dp8a were identical to those of R4 in dpόa indicating a reducing end sequence of IdoA(2S)-GlcNS-IdoA-GlcNAc.

The complete sequence of dp8a is therefore: ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-GlcNS-IdoA-GlcNAc.

This again is compatible with the disaccharide analysis (Table 1).

The presence of U4 and U6 fragments after nitrous acid depolymerisation are further confirmation that the sequence has three internal N-sulphated units. The elution position of U4 in dp8a was identical to that of U4 in dpόa and this would be predicted from the sequences of dp6a,dp8a. Although not strictly necessary to sequence an unknown oligosaccharides, the predictable nature of these "common fragments" did prove useful, particularly in reducing the number of peaks to be classified within a set of profiles.

EXAMPLE 5 Major decasaccharide 10a was subjected to partial depolymerisation and separation as described in Example 3. The profiles for the sequencing of the major decasaccharide 10a are shown in Figure 8. The profiles in panels B-D were obtained by digesting aliquots of the fragments (having profile A) with iduronate-2-sulphatase (B), iduronidase (C), or both enzymes sequentially (D). Once again U4 and U6 are clearly identified by their resistance to the exoenzymes. Fragments R4, R6 and M4 all move with iduronate-2-sulphatase, but not with iduronidase alone. It is not possible to accurately identify either R8 or U8. Fragments U6 and R6, being derived from opposite ends of the dp 10 sequence, overlap in the central disaccharide repeat and this fact essentially solves the sequence. Other identified fragments (e.g. R4 and U4) are all compatible with the following final sequence

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-

GlcNS-IdoA(2S)-GlcNS-IdoA-GlcNAc

EXAMPLE 6

More complex species are also easily solved using this technique. When the products of partial nitrous acid scission of octasaccharide 8d were separated by HPLC, the presence of 6-O-sulphation was indicated by a peak corresponding to the disaccharide IdoA(2S)-aMan(6S) (Figure 9). Therefore an additional enzyme digestion step was included in this analysis, combining 6-sulphatase (used at 0-096μU per digest), iduronate-2-sulphatase and iduronidase. Thus the 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). The results are shown in Table 5 as follows: TABLE 5 a b c d e f g h

ΔUA- GIcNS0₃- IdoA(2S)- GlcNS0₃(6S)- IdoA(2S)- GICNSO3 -IdoA- GlcNAc 8d

ΔUA- GICNSO3- IdoA(2S)- GlcNSθ3(6S)- IdoA(2S)- aMan U6

IdoA(2S)- G!cNS03(6S)- IdoA(2S)- GICNSO3 -IdoA- GlcNAc R6

IdoA(2S)- GICNSO3 -IdoA- GlcNAc R4

IdoA- GlcNS0₃(6S)- IdoA(2S)- GIcNS0₃ -IdoA- GlcN Ac R6'

GlcNSθ3(6S)- IdoA(2S)- GICNSO3 -IdoA- GlcNAc R6^M

GICNSO3- IdoA(2S)- GICNSO3 -IdoA- GlcNAc R6'" Once again, it is easy to identify fragments that are not affected by any of the enzyme treatments (i.e. U6 and the intact dp8) as well as one tetrasaccharide (R4; the same as was seen in 6a and 8a) which shifts with the iduronate-2-sulphatase alone, and further with iduronate-2-sulphatase plus iduronidase. An additional peak is also observed which elutes at fraction 190 (R6), and which shifts with iduronate-2- sulphatase to fraction 150 (R). This latter peak displays only a small shift when subsequently treated with iduronidase (fraction 148, R"), but the combination of these two enzymes must be removing the hexuronate as the subsequent addition of 6- sulphatase causes a further significant shift to fraction 98 (R6'"). Fragment R6 therefore contains a non-reducing terminal sequence of IdoA(2S)-GlcNS(6S) and is a reducing end fragment of dp8d. The presence of R6 after nitrous acid scission shows that the unsaturated disaccharide of dp8d is N-sulphated. Confirmation that the unsaturated uronic acid lacks 2-sulphation comes from the presence of the nonsulphated disaccharide peak in all of the profiles (fraction 18), specifically this peak is still observed after the combined iduronate-2-sulphatase and iduronidase digest. Combining the sequence data of R6 with the presence of R4, the sequence of dp8d is: ΔUA-GlcNS-IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS-IdoA-GlcNAc.

This same principle of observing the results of digesting partial nitrous acid- generated fragments with exo-enzymes was therefore used to deduce the sequences of all the oligosaccharides labelled in Figure 5, panels A and B, as well as two decasaccharides from Figure 5, panel C (see Table 6).

TABLE 6

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA-GlcNAc 6a

ΔUA-GlcNS-IdoA(2S)-GlcNS-GlcA-GlcNAc 6b ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-GlcNAc 6c

ΔUA-GlcNS-IdoA(2S)-GlcNS(6S)-IdoA-GlcNAc 6d

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-GlcNS-IdoA-GlcNAc 8a

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-GlcNS-GlcA-GlcNAc 8b

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-GlcNAc 8c* ΔUA-GlcNS-IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS-IdoA-GlcNAc 8d

ΔUA-GlcNS-IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS-GlcA-GlcNAc 8e

ΔUA-GlcNS-IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS-IdoA(2S)-GlcNAc 8f

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)- GlcNS-IdoA(2S)-GlcNS-IdoA-GlcNAc 10a

ΔUA-GlcNS-IdoA(2S)-GlcNS-IdoA(2S)-

GlcNS(6S)-IdoA(2S)-GlcNS-IdoA-GlcNAc 10c

In each case, the ability to match common fragments between analyses (e.g.

R4 in 6a, 8a, and 1 Oa), and to independently ascertain the disaccharide composition by multi-heparinase digestion, helped to confirm the sequences.

As will be seen, 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.

Although 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.

Claims

1. A method of analysing and sequencing saccharide material comprising saccharide chains which contain more than three monosaccharide units interconnected through glycosidic linkages that are not all identical, wherein selected exoenzymes comprising exoglycosidases of known specificity that cleave only particular glycosidic linkages at the non-reducing end of saccharide chains and chain fragments are used to obtain sequence information, said method comprising the sequential steps of :

(a) subjecting said saccharide material to partial depolymerisation by controlled treatment with a selective scission reagent that acts in accordance with a known predetermined linkage specificity as an endoglycosidase to cleave a proportion of susceptible internal glycosidic linkages, that is, susceptible glycosidic linkages spaced from the non- reducing end of the saccharide chains, 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,

(b) treating a selected sample or samples of said mixed set of saccharide chains and chain fragments with said exoenzymes, either singly or in combination in accordance with a predetermined strategy, to an extent sufficient to obtain complete digestion and cleave susceptible linkages at the non-reducing end of all the saccharide chains and chain fragments, and then (c) analysing said sample or samples to detect the various saccharide chain fragments generated by the cleavage treatments which are present therein, and obtaining, collectively from the results of said analysis, information enabling the monosaccharide sequence in the original saccharide material to be at least partially deduced; wherein substantially all of the saccharide chains and chain fragments produced by step (a) are labelled.

2. A method as claimed in Claim 1 wherein saccharide chains and chain fragments which are the product of step (a) are labelled on substantially every monosaccharide residue.

3. A method as claimed in Claim 1 wherein saccharide chains and chain fragments which are the product of step (a) are labelled on substantially every disaccharide unit or residue.

4. A method as claimed in Claim 1, 2 or 3 in which step (a) comprises the additional step of labelling the saccharide chains and chain fragments produced by the partial depolymerisation.

5. A method as claimed in Claim 1 in which step (a) comprises the additional step of labelling the reducing ends of the saccharide chains and chain fragments produced by the partial depolymerisation.

6. A method as claimed in any one of the preceding claims wherein said saccharide chains and chain fragments are labelled by a detectable label or tag which is selected from: a radiochemical labelling agent, a fluorescent labelling agent, and a colorimetrically detectable labelling agent.

7. A method as claimed in Claim 6 wherein the detectable label or tag is a radiochemical labelling agent.

8. A method as claimed in Claim 2 wherein the detectable label or tag is a radiochemical labelling agent and wherein the saccharide material is derived from cells or tissues grown in culture wherein the radiochemical labelling agent is present in the culture medium.

9. A method as claimed in Claim 8 wherein the radiochemical labelling agent is H-glucosamine and/or ³²S-sulphate.

10. A method as claimed in any one of the preceding claims wherein the selected exoenzymes also include specific exosulphatases for bringing about a controlled selective removal of sulphate groups from monosaccharide residues at the non- reducing end of said saccharide chains or fragments of said chains.

1 1. A method as claimed in any one of the preceding claims wherein the selective scission reagent used in step (a) for bringing about the controlled partial depolymerisation of the saccharide material is selected from nitrous acid and endoglycosidase enzymes.

12. A method as claimed in Claim 11, wherein said selective scission reagent is nitrous acid which is used under low pH conditions such that it acts specifically to cleave hexosaminidic linkages that link an N-sulphated amino sugar to a hexuronate residue but not hexosaminidic linkages that link an N-acetylated amino sugar to a hexuronate residue.

13. A method as claimed in any one of the preceding claims wherein the controlled depolymerisation step is carried out by treating separate samples of the saccharide material with said selective scission reagent for different periods of time and then pooling the products to provide the mixed set of chain fragments for use in step (b), thereby to assist in ensuring that the lengths of the saccharide chain fragments in said mixed set of chain fragments used in step (b) are distributed throughout the range extending from full length undegraded chains to minimum length chain fragments lacking in further internal glycosidic linkages cleavable by said selective scission reagent.

14. A method as claimed in any one of the preceding claims in which saccharide chain fragments produced by the cleavage treatments are separated according to their length and composition in carrying out the analysis of step (c).

15. A method as claimed in any one of Claims 1 to 13 in which a sample of the mixed set of saccharide chain fragments produced by the partial depolymerisation of the saccharide material in step (a) is subjected without exoenzyme treatment to a separation procedure to separate said chain fragments according to length, and other samples are also subjected, separately, to the same said separation procedure to separate the saccharide chain fragments therein according to length and composition after each of these other samples has been treated with a different exoenzyme or combination of exoenzymes in step (b).

16. A method as claimed in Claim 14 or 15, wherein the separated saccharide chain fragments are detected by detecting a label or tag carried thereby.

17. A method as claimed in any one of Claims 14, 15 or 16, wherein said separation of the saccharide chain fragments is effected by an electrophoretic separation technique or high performance liquid chromatography whereby the saccharide fragments are separated according to differences in length and composition.

18. A method as claimed in Claim 17 wherein separate samples of the mixed set of saccharide chain fragments from step (a) are treated with different said exoenzymes (singly or in combination) in carrying out step (b) and are subjected simultaneously in step (c) to electrophoretic separation in different tracks of an electrophoresis gel, thereby providing a migration banding pattern representative of the different gel mobilities of the separated fragments that have differences in length and/or composition.

19. A method as claimed in Claim 18 wherein the electrophoretic separation is carried out by gradient polyacrylamide gel electrophoresis (gradient PAGE).

20. A method as claimed in Claim 18 or 19 in which the separated saccharide chain fragments are transferred by an electrotransfer technique to a nylon membrane after termination of the electrophoresis.

21. A method as claimed in any one of the preceding claims wherein the labelled or saccharide chains of the saccharide material or the fragments thereof provide in the analysis of step (c) a visually-detectable pattern which, in conjunction with the known specificity of the exoenzymes used in step (b), gives sequence information directly by deduction.

22. A method as claimed in any one of the preceding claims in which the saccharide material is composed of saccharide chains containing amino sugar residues and sulphated monosaccharide units, for example glycosaminoglycan saccharide chains, and the exoenzymes used in step (b) include at least some of the following: a glucuronidase, an iduronidase, an N-acetylglucosaminidase, an iduronate-2- sulphatase, a glucuronate-2-sulphatase, a glucosamine-6-sulphatase (e.g. N- acetylglucosamine-6-sulphate sulphatase), a glucosamine-3 -sulphatase and a sulphamate sulphohydrolase (sulphamidase).

23. A method of analysing saccharide material in order to determine the sequence of monosacchardide units in saccharide chains thereof, carried out substantially as herein described with reference to the accompanying drawings.