ISOLATION OF CARBOHYDRATES FROM CELL TISSUE

Abstract: A process for isolating the carbohydrate fraction (oligosaccharides) from cell tissue with degradation of the glycoconjugates, the cell tissue being treated with a reagent containing or consisting of trifluoroacetic acid anhydride, and the carbohydrates being released under conversion to trifluoracetates, which are optionally detrifluoroacetylated. Starting from erytrocyte membranes oligosaccharides possessing blood group specificity can be recovered, and such oligosaccharides are useful for blood group determination.

Description: TITLE OF INVENTION; ISOLATION OF CARBOHYDRATES FROM CELL TISSUE. TECHNICAL FIELD; The present invention relates to a process of isolating from different types of cell tissue carbo¬ hydrates mainly consisting of oligosaccharides with degradation of glycoconjugates present in the cell tissue, said glycoconjugate being a common term for glycoproteins, proteoglycanes and glycolipids. Cell tissue is a complex material consisting inter alia of so-called glycoconjugates, which are constituted by complex structures composed of carbohydrates, proteins and lipids. The three main types of glycoconjugates are glycoproteins, proteoglycanates and glycolipids. In the glycoproteins the carbohydrate chains are mostly N-glyco- sidically bound to asparagine or O-glycosidically to serine, treonine, hydroxylysine or hydrόxyproline. The carbohydrate chains of the proteoglycanes have similar bonds to the protein part. In the glycolipids, finally, -the carbohydrate part is O-glycosidically bound and .is present as glycosyl ceramides in man and animals. BACKGROUND ART: The carbohydrate part of cell membrane glycoconju¬ gates has been found to play an essential role in several biological functions including cell-cell interactions, as receptors for antibodies, enzymes, hormones, viruses and toxines. In order to determine the structure and to study the biological activity of the carbohydrate part of glycoconjugates in cell membranes it is desirable to release the oligosaccharide chains in a form as intact as possible. Such a procedure would be preferred compared to the almost impossible task cf isolating glycoconju¬ gates in a pure form. SUMMARY OF THE INVENTION; Thus, the present invention has for its purpose to provide a process for releasing the carbohydrate part from the glycoconjugates in cell tissue while maintaining the carbohydrate chains in a more or less intact state. More in particular, the invention provides for a process for isolating the carbohydrate fraction from glycolipids of cell tissue by specifically cleaving the glycosidic bond between the carbohydrate part and the ceramide residue. Moreover, in another aspect of the invention, there is provided a process of isolating, from complex carbohydrate structures of cell tissue (glycolipids, proteoglycanes, and glycoprotein), oligo¬ saccharides bound to a di-substituted 2-acetamido-2-- -deoxyhexose by specifically cleaving the 2-acetamido-2- -deoxyhexosepyranosidic bond and then by elimination reactions releasing the ligosaccharide substituents. The present invention is based on technique residing in treating the cell tissue with a reactant containing or consisting of trifluoroacetic acid anhydride. By this treatment, which can be termed trifluoroacetolysis, the oligosaccharide chains are released from both glyco¬ proteins and glycolipids with simultaneous degradation of proteins and lipids, whereby the oligosaccharides in the form of their trifluoroacetates can be separated and recovered. As a reactant for treating the cell tissue there may be used trifluoroacetic acid anhydride solely or trifluoroacetic acid anhydride in combination with tri- fluoroacetic acid, the latter constituting up to about 90 % by volume of the reactant mixture. A preferred reactant is one wherein trifluoroacetic acid anhydride and trifluoroacetic acid are present in about equal proportions based on volume. The treatment with the reactant is preferably carried out at an increased t-emperature and at an increased pressure. A preferred temperature range is 8θ-110°C, for example about 100°C. The overpressure in the latter case is about atms. In a preferred embodiment of the process according to the present invention the cell tissue is constituted by erytrocyte membranes from which can be recovered oligosaccharides possessing blood group specificity. Such blood group specific oligosaccharides recovered in accordance with the technique of this invention can be coupled to carriers with antigenicity, and conjugates obtained thereby may then be used for immunisation for the purpose of obtaining specific antibodies. An alterna¬ tive use of isolated oligosaccharides is coupling of said saccharides to carriers for the preparation of immunosorbent columns which can be used. for purification and isolation of specific antibodies. The specific anti¬ bodies made can be used in reactants for blood group determination and for other medicinal purposes. The reaction mixture obtained when treating the cell tissue with the reactant is suitably evaporized to dryness, and the residue vaporization is distributed between water and a water-immiscible solvent, the aqueous phase containing the carbohydrate fraction being recovered and the solvent phase containing inter alia protein residues and lipid residues being discarded. As a water-immiscible solvent there may be used for example ether or methylene chloride. As a pretreatment for the trifluoroacetolysis the erytrocyte membranes are suitably homogenized and suspended in water, whereafter lyop ilisation is carried out before the treatment with the reactant. The oligosaccharides recovered by the process according to this invention may suitably be purified by gelchromatography and preparative paper chromatography. In this manner there may be obtained from blood of the 'BUREAU OMPI 3&aϊτιøg^ blood group A the oligosaccharide: -GalNAcp-(1- )-Gal i (A-tri) !__ I α-Fucp -Galp-(l- )-β-Galp-(l- )-Glc or -GalNAcp-(l-3)-α-Galp-(l- )-β-Galp-(l- )-Glc. From blood of the blood group B there is obtained in addition to the two last-mentioned instead of A-tri the oligosaccharide: -Gal-(1-3)-Gal T (B-tri) α-Fuep From blood of the blood group 0 there is obtained in the same manner the oligosaccharide: α-Fucp-(1-2)-Gal instead of A-tri. All the oligosaccharides mentioned under A- and B-blood are obtained from AB-blood. With regard to the preferred application of the technique according to this invention, namely release and recovery of oligosaccharides from erytrocyte membranes which show blood group specificity, one may thus observe that up to the practical use four main steps are part of the procedure, namely the following: a. Isolation of blood group specific oligo¬ saccharides from erytrocyte membranes; b. Chemical modification of isolated oligo¬ saccharide for coupling to an antigenic carrier or a carrier for the preparation of immunosorbent. c. Immunisation of animals, alternatively pre¬ paration of antibodies by means of hybridom technique. d. Purification of antibodies. The present invention will now be described by non-limiting examples which are based upon the preferred application of the invention for the preparation of oligosaccharides carrying blood group antigenicity from erytrocyte membranes. EXAMPLES a. Preparation of oligosaccharides. Erytrocytes from discarded blood of blood groups A, B, AB and 0 are lysed and the membranes are isolated by means of centrifugation. In this way about 10 grams of membranes are obtained from one liter of blood. The membranes are treated according to the process of Dodge, J.T., Mitchell, C, and Hanahan, D.J. (1963), Arch. Biochem. Biophys. 100, 119-130, i.e. homogenised, suspended in water and lyophilised. To 50 grams of the lyophilised material there are added trifluoro acetic acid anhydride (1.25 liter) and trifluoro acetic acid (1.25 liter), and the reaction mixture is heated at about 100°C in a tube of acid- -resistant stainless steel at an overpressure of about atms for a period of time of about 48 hours. After this treatment the reaction mixture is cooled and evaporized to dryness, there being obtained a dark to black-coloured residue. To said residue there is added methanol (700 ml), and the mixture is evaporised to dryness. The residue is then diluted with 50 % aqueous solution of acetic acid (1000 ml), and is allowed to star.ά at room temperature for about l8 hours. The reaction mixture is filtered with a glass filter and evaporised to dryness. The residue obtained is distributed between water and diethyl ether. The ether phase is washed 4 times with water, and the combined wash solutions are washed with diethyl ether 4 times. The aqueous solution obtained is yellow-coloured and contains the released oligcsaccharides. The oligosaccharides obtained are in such form that the i-acetyl functions have been converted to N-trifluoroacetyl groups. The oligosaccharides obtained can be purified* by gel chromatography followed by a preparative paper chromatography, and the purification procedure is continuously surveyed with gas chromatography- -mass spectrometry. The following oligosaccharides are obtained in the present example by treatment of blood originating from blood groups A, B, AB and 0 with varying blood groups within the P-system: A-blood Speci- Yield, ficity mg/g membrane Oligosaccharide α-GalNAcp-(l-3 A 1 α-Fucp α-Galp-(l-4)-β-Galp-(l-4)-Glc ■ p k 1 α-GallRcp-(l-3)-α-Galp-(l-4)-β-Ga_p-(l-4)-Glc. P. 10 (globoside) B-blood α-Gal-(1-3)-Gal B 1 "≥ i α-Fucp trihexoside p 1 globoside P 10 AB-blood All stated under A-blood and B-blood. 0-blood α-Fucp- (1-2)-Gal 0(H) 1 trihexoside p 1 globoside p 10 b. Chemical modification of isolated oligosaccharides. In order that the oligosaccharides shall be capable of coupling to carriers of different kinds they should be chemically modified. Such modification can be per¬ formed by means of known technique. Before the chemical modification it is suitable to de-N-trifluoroacetylate the oligosaccharides, which is suitably carried out in a basic environment at a pH of about 10-12 and at room temperatures for a period of time of about 20 hours. This results in the formation of a ino groups, which can be N-acetylized to reform the N-acetyl functions. The procedure for modifying isolated oligosaccharides * is exemplified below for a B-active trisaαcharide, namely α-Galp-(l-3)-LL-Fuep- (l-2)-*J -Gal diagrammatically in the following way: α-Galp- (1-3)-Gal 1. Acetsrlation α-Galp- (l-3)-β-Galp-0CH 2 CH 2 CH 2 N.H 2 £ 2. HBr/AcOH " " * I I i" 3. H0CH 2 CH 2 CH 2 N0 2 ^ L-Fucp (Koenigs-Knorr) L-Fuep 4. Hydrogenation The above reaction steps can be carried through with a total yield of at least about 50 %. The aliphatic amino groups formed by the de-N-tri¬ fluoroacetylation can be used for coupling the modified oligosaccharide to different carrier materials. c. Immunisation of animals. The modified oligosaccharides can be coupled to proteins, which can be constituted by albumen or edestine, and possibly to lipid A (from lipopolysaccharide) for the purpose of obtaining a high yield of 7M-antibodies. The conjugates obtained are then used for immunising animals, for example rabbit or horse, or for the production of antibodies by hybridom technique (Kohler, G. and Mil- stein, C, 1975, Nature (London) 256, pp. 495-497; Kohler G., and Milstein, C, 1976, Eur.J.Immunol. 6 pp. 5H-519) • The antisera thereby obtained are then serologically used in connection with known blood classi- fication methods. "BU EATΓ OMPl ^ NATIO^ Purification of antibodies. Chemically modified blood group active oligo¬ saccharides prepared according to the above are coupled to a suitable carrier material, for example Sephadex, and then sera from immunised animals are passed through an immunosorbent column formed by the treated Sephadex. Antibodies having specificity vis-a-vis coupled oligo¬ saccharide adheres by reaction with same, whereas mate¬ rials of no interest are washed away. The specific antibodies adhering to the column by reaction with coupled oligosaccharides are then desorbed, and antibody solutions of unambiguously defined specificity are obtained. Purified specific antibodies ar^ then tested for specificity for control and may then be used for blood group determination by hemagglutination- technique. It should be observed that the present invention is not delimited to the exemplary application for preparation of blood group active oligosaccharides from erytrocyte membranes as described above. The invention is applicable to all type ' s of cell tissue for recovery and isolation • of oligosaccharides for different purposes. As further examples there may be mentioned epithelium tissue (intestinal mucous membrane, urinary tract epi- thelium, lung epithelium etc.), wherein isolated oligo¬ saccharides can be of diagnostic importance. In this disclosure the abbreviations used in the structural formulae have the following meaning: GalNAcp = 2-acetamido-2-deoxy-D-galactopyranosyl Gal = p-galactose Fucp = 6-deoxy-L-galactose Galp = D-galactopyranosyl Glc = D-glucose

Claims: CLAIMS: 1. A process for isolating the carbohydrate fraction (oligosaccharides) from cell tissue with degradation of the glycoconjugates, characterized thereby that the cell tissue is treated with a reagent containing or consisting of trifluoroacetic acid anhydride, the carbohydrates being released under con¬ version to trifluoroacetates, which are optionally detri- fluoroacetylized. 2. A process according to claim 1, characterized thereby that the treatment with the reagent is performed at an increased temperature and at an increased pressure, for example about 100°C and about 4 atms. overpressure. 3. A process according to claim 1 or 2, charact¬ erized thereby that the treatment is performed with a reagent consisting of trifluoroacetic acid anhydride and up to about 90 % by volume of trifluoroacetic acid. 4. A process according to any of the preceding claims, characterized thereby that the reaction mixture obtained by treatment of the cell tissue with the reactant is evaporized to dryness and that the evaporization residue is distributed between water and a water-immiscible solvent, the aqueous phase containing the carbohydrate fraction being recovered and the solvent phase being discarded. 5- A process according to any of the preceding claims, characterized thereby that the cell tissue is constituted by erytrocyte membranes, oligosaccharides being recovered which show blood group specificity. 6. A process according to claim _>, characterized thereby that the erytrocyte membranes are homogenized, suspended in water and lyophilized before the treatment with the reagent. tfϋR E Aϊr OMPl _*&_«& 7. A process according to any of the preceding claims, characterized thereby that oligosaccharides recovered are purified by gel chromatography followed by paper chromatography. 8. A process according to claim " , characterized thereby that starting from erytrocyte membranes from blood of the A-group there is obtained the oligosaccha¬ ride: α-GalNAcp-(l-3)-Gal I W _ _ α-Fucp α-Galp-(l-4)-β-Galp-(l-4)-Glc or α-GalNAcp-(l-3)-α-Galp-(l-4)-β-Galp-(l-4)-Glc. 9- process according to claim 7, characterized thereby that starting from erytrocyte membranes from blood of the B-group there is obtained the oligo¬ saccharide: α-Fucp α-Galp-(1-4)-β-Galp-(l-4)-Glc or α-GalNAcp-(l-3)-α-Galp-(l-4)-β-Galp-(l-4)-Glc. 10. A process according to claim 7 * characterized thereby that starting from erytrocyte membranes from blood of the 0-group there is obtained the oligo¬ saccharide: α-Fucp- (l-2)-Gal, α-Galp-(l-4)-β-Galp-(l-4)-Glc or α-GalNAcp-(l-3)-α-Galp-(l-4)-β-Galp-(l-4)-Glc. 11. A process according to claim 7, characterized thereby that starting from erytrocyte membranes from blood of the AB-group there Is obtained the oligo- " ■ UR ATT - OMPI ^SNATlp saccharide: α-GalNAcp-(1-3)-Gal i α-Gal-(1-3)-Gal I -b I α-F cp α-Galp-(1-4)-β-Galp- (1-4)-Glc or α-GalNAcp-(l-3)- -Galp-(l-4)-β-Galp-(l-4)-Glc.

Inventors: SVENSSON S
Applicants: SVENSSON S
Application year: 1981
Main classification: A61K31
IPC codes: A61K31, A61K35, C07H1, C07H3, C07K16, G01N33