Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving glucose or galactose
Reexamination Certificate
1997-10-20
2003-07-08
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving glucose or galactose
C536S124000
Reexamination Certificate
active
06589757
ABSTRACT:
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 containing numerous amino sugar residues such as, for example, are found in glycosaminoglycans (GAG's) which include the biologically important polysaccharides, heparan sulphate (HS) and heparin.
BACKGROUND
Heparan sulphate (HS) and heparin are chemically-related linear glycosaminoglycans (GAG's) composed of alternate &agr;,&bgr;-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 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 one aspect the invention may be regarded as being 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. Although the invention may be 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 GAG's and different saccharides, including the saccharide component of glycoproteins.
Use of exoenzymes, in particular exoglycosidsases, for removal of terminal sugar residues at the non-reducing end of saccharide chains has previously been proposed in connection with methods for sequencing such chains, for instance in WO 92/02816 and in WO 92/19974 and WO 92/19768. However, in these 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 is 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 is 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. In all these prior art methods the sequencing information is obtained and presented in a different manner to that in the present invention.
An acknowledgement is also made of a paper by Kyung-Bok Lee et al, Carbohydrate Research, 214 (1991), 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 defined in the claims relating to the present invention.
More specifically, the present invention broadly provides 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 and which each include a referencing feature at their reducing end, wherein selected exoenzymes comprising exoglycosidases of known specificity that cleave only particular glycosidic linkages at the non-reducing end of saccharide chains are used to obtain sequence information, said method being characterised in that it comprises 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 then
(c) analysing said sample or samples to detect the various saccharide chain fragments generated by the cleavage treatments which are present therein and which have a reducing end derived from the reducing end of the corresponding chain in the original saccharide material, 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.
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 at their reducing ends in order to introduce the reducing end referencing feature for providing a common reference point or reading frame to which the monosaccharide sequence can be related and for facilitating, during analysis, detection of chain fragments having a reducing end derived from the reducing end of the corresponding chains in the original saccharide material. This end referencing feature is conveniently provided by selectively labelling or tagging the monosaccharide units at the reducing ends of the saccharide chains, using for example radiochemical, fluorescent, biotin or other calorimetrically detectable labelling means.
If low pH nitrous acid is used for carrying out the partial depolymerisation of the saccharide material as hereinafter described, a presently preferred fluorescent labelling agent is anthranilic acid as referred to in more detail later. However, if a selective scission reagent other than nitrous acid is used for bringing about the partial depolymerisation, e.g. an endoglycosidase enzyme, an aminocoumarin hydrazide, e.g. 7-amino4-methylcoumarin-3-acetyl hydrazide, may be preferred for providing a fluorescent labelling agent having a relatively high labelling efficiency. For use as a radiochemical labelling agent tritiated borohydride may be used.
In an alternative but usually less preferred technique for providing end-referenced saccharide chains or chain fragments, the chains may be immobilized by
Gallagher John Thomas
Hopwood John Joseph
Turnbull Jeremy Ewan
Cancer Research Technology Limited
Gitomer Ralph
Pillsbury & Winthrop LLP
LandOfFree
Sequence analysis of saccharide material does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Sequence analysis of saccharide material, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Sequence analysis of saccharide material will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3060835