WO2002092845A2 - Method for determining enzymatic activity of glycosyl transferase - Google Patents

Abstract

The invention relates to a method for determining the enzymatic activity of glycosyl transferase in a sample, wherein the sample is reacted with a glycosyl donor and a glycosyl receptor immobilized on a carrier, especially a cellulose membrane, wherein a transferase product is determined in the presence of a enzymatic glycosyl transferase activity and the carrier is a cellulose membrane.

Description

 Method for the detection of the enzymatic activity of glycosyltransferases

The invention relates to a method for detecting the enzymatic glycosyltransferase activity in a sample, a method for determining the glycosyltransferase activity pattern of a sample, a method for determining the substrate specificity of a glycosyltransferase, use of the above-mentioned methods in the diagnosis of Diseases, a method for identifying a compound influencing the expression of a glycosyltransferase and a method for identifying a compound influencing the enzymatic activity of a glycosyltransferase and a kit for the detection of the enzymatic activity of a glycosyltransferase.

Glycosyltransferases transfer activated mono- or oligosaccharides from glycosyl donors to glycosyl acceptors. Mono-, oligo- or polysaccharides, peptides, proteins or lipids, as well as their combinations, which are then present as glycoconjugates, can serve as acceptors. Glycoconjugates play a key role in many biological processes. They act as receptor sites for enzymes, hormones, antibodies, lectins, toxins, and for microorganisms such as viruses and bacteria. The involvement of glycoconjugates in the control of cell functions such as cell growth, cell-cell recognition, expression of antigens, cell adhesion, infection and cell differentiation is particularly important physiologically and pathophysiologically. The growth-dependent pattern of glycosylation of the glycoproteins is mainly determined by the activity and specificity of the glycosyltransferases located in the Golgi apparatus.

Changes in the activity and expression of specific glycosyltransferases are closely related to tumor development and other disease states. For example, the changed composition of the cell surface oligosaccharides is directly related to the degree of transformation and metastasis formation (Dennis, JW, Laferte, S., Waghorne, C, Breitman, ML, Kerbel, RS, Science 1987, 236, 582). Malignant changes in mouse and human cells are generally associated with the expression of more complex N-glycosidic structures and a higher polylactosamine content (Warren, L., Bück, CA, Tuszynski, GP, Biochem. Biophys. Ada. 1978, 516, 97) , The latest studies show the branching and the polylactosamine content of the O-oligosaccharides with the tumor development and metastasis formation in connection (Yousefi, S., Higgins, E., Daoling, Z., Pollex-Kxuger, A., Hindsgaul, O. and Dennis, JW, J. Biol. Chem. 1991, 266, 1772).

The glycosyltransferases are classified according to the nucleotide glycosyl donors (activated glycosyl donors of uridine diphosphate (UDP-Gal, UDP-GlcNAc, UDP-Glc, UDP-GalNAc, UDP-Xyl, UDP-GlcA), guanosine diphosphate (GDP-Fuc , GDP-Man), and cytidine monophosphate (CMP-neuraminic acid or CMP-Neu5Ac), the type of glycosidic bond (α or ß), and the hydroxyl group of the acceptor involved, to which the monosaccharide is transferred, with few exceptions, transferases catalyze highly Regio- and stereo-selective, which is why about 200 different glycosyltransferases must exist in order to enable all known oligosaccharide structures (Paulson, J. C, and Colley, KJ, J. Biol. Chem. 1989, 264, 17615) The acceptor substrate is based on only a few hydroxyl groups, the so-called “polar key groups.” This has already been possible in numerous protein-carbohydrate interactions such as with lectins, antibodies, glycosidases and oxidas s are shown (Lemieux, R. U., Acc. Chem. Res. 1996, 29, 373).

The αl, 3-galactosyltransferase (αl, 3-GalT, EC 2.4.1.151), which catalyzes the transfer of galactose from UDP-Gal to the 3-OH group of galactose in structures containing Galßl- »4GlcNAc-R (Fig. 1) and thus responsible for the formation of Galαl- »3Galßl-» 4GlcNAc epitopes that occur on glycoproteins and glycolipids has recently become increasingly important (Joziasse, DH, Shaper, NL, Salyer, LS, Van den Eijnden, DH, van der Spoel, A.C, Shaper, JH, Eur. J. Biochem. 1990, 191, 75; Fang, J., Li, J., Chen, X., Zhang, Y., Wang, J., Guo Z., Zhang, W., Yu, L., Brew, K., Wang, PG, J. Am. Chem. Soc. 1998, 120, 6635; Sujino, K., Malet, C, Hindsgaul , O., Palcic, MM, Carbohydr. Res. 1998, 305, 483). The Galαl— »3Galßl—> 4GlcNAc epitope is the most important antigen which is responsible for the acute rejection of transplants (Galili, U., mmunol. Today 1993, 14, 480; Joziasse, DH, Oriol, R., Biochim Biophys. Acta 1999, 1455, 403). Recombinant αl, 3-GalT and a fusion protein consisting of UDP-Gal epimerase and αl, 3-GalT are available for synthetic use (Wang J.-Q., Chen, X., Zhang, W., Zacharek, S., Chen, Y., Wang, PG, J. Am. Chem. Soc. 1999, 121, 8174). In contrast to the β1, 4-GalT, the αl, 3-GalT is not able to transfer UDP-Glc, -GalNAc, -GlcNAc and -glucuronic acid residues (Stults, CLM, Macher, BA, Bhatti, R ., Srivastava, OP, Hindsgaul, O., Glycobiology 1999, 9, 661). Other acceptors such as Galßl- »3GlcNAc, Galßl → 4Glc (Sujino, K., Malet, C, Hindsgaul, O., Palcic, MM, Carbohydr. Res. 1998, 305, 483; Blanken, WM, van den Eijnden, DH , J. Biol. Chem. 1985, 260, 12927) or polymer-bound acceptors are accepted by the α1, 3-GalT (Wang J.-Q., Chen, X., Zhang, W., Zacharek, S., Chen , Y., Wang, PG, J. Am. Chem. Soc. 1999, 121, 8174).

Many of the antigenic oligosaccharides found on cell surfaces, such as blood group antigens, are fucosylated. In addition to sialylation, fucosylation is often the last step in the in vivo modification of oligosaccharides. Fucosylated cell surface oligosaccharides are involved in a large number of intercellular recognition processes (Macher, BA, Holmes, EH, Swiedler, SJ, Stults, C. L, Srnka, CA, Glycobiology 1991, 1, 577; Hakomori, S.-L, Adv. Cancer. Res. 1989, 52, 257; Feizi, T., Nature 1985, 314, 53). The α1, 3/4-fucosyltransferases catalyze the transfer of fucose residues from GDP-fucose to the 3- or 4-OH group of a variety of ß-D-galactose-containing structures to form the Le ^a and Le ^x blood group antigens (Macher , BA, Holmes, EH, Swiedler, SJ, Stults, C. I, Srnka, CA, Glycobiology 1991, 1, 577; Natsuka, S., Löwe, JB Curr. Opin. Struct. Biol. 1994, 4, 683; Oriol, R., Mollicone, R., Cailleau, A., Balanzino, L., Breton, C, Glycobiology 1999, 9, 323; Watkins, WM, Carbohydr. Res. 1986, 149, 1). Breast milk is by far the most important source for the isolation of the preparative α1, 3/4 fucosyltransferases (Eppenberger-Castori, S., Lotscher, H., Finne, J., Glycoconjugate J. 1989 6, 101). Both the α1, 3/4-fucosyltransferase and the 1,3, fucosyltransferase occur in breast milk. Five different human αl, 3/4 fucosyltransferases, FucT III to FucT VII, have already been cloned. The in vivo selectivity of these human fucosyltransferases, which were expressed in BHK cells, has already been elucidated (Grabenhorst, E., Nimtz, M., Costa, J. and Conradt, HS, J. Biol. Chem. 1998, 273, 30985 ). The ocl, 3/4-FucT (EC 2.4.1.65) obtained from breast milk is an exception to the "one enzyme per bond" hypothesis, since both the transfer of fucose to Galßl → θGlcNAc (type I) and Galßl → 4GlcNAc ( Type II) structures are catalyzed to produce compounds of the Le ^a or Le ^x type, as is also shown in FIG. 2. The fucosyltransferase FucT V is very similar to the 1,3 / 4-FucT from breast milk, since both type I and type U acceptors are used as substrates (cf. FIG. 2). FucT V is an important industrial enzyme that was already used in the industrial production of Sialyl Lewis ". The sialyltransferases catalyze the transfer of N-acetylneuraminic acid from CMP-Neu5Ac to hydroxyl groups near the non-reducing end of oligosaccharide chains of the glycoproteins or glycolipids (Paulson, J.C. and Colley, KJ, J. Biol. Chem. 1989, 264, 17615 ; Harduin-Lepers, A., Recchi, M.-A., Delannoy, P., Glycobiology 1995, 5, 741; Tsuji, S., Datta, AK, Paulson, J. C, Glycobiology 1996, 6, v) , Neuraminic acid residues on cell surfaces play an important role as antigenic determinants, as ligands for cellular adhesion processes and as receptors for viruses and bacteria (Keim, S., Schauer, R., Int. Rev. Cytol. 1997, 175, 137). To date, 16 different sialyltransferases have been identified in mammals, as well as several bacterial and viral sialyltransferases. A distinction can be made between α2.3, α2.6 and α2.8 sialyltransferases. A systematic classification of the sialyltransferases based on the regiochemistry of the sialylated reaction products and the acceptor substrates has been proposed (Tsuji, S., Datta, AK, Paulson, J. C, Glycobiology 1996, 6, v). Two different recombinant α2,3-sialyltransferases and an α2,6-sialyltransferase from rat liver are already commercially available in 100 mU quantities for preparative work. α2,3-SialT (α2,3-SialT, EC 2.4.99.6, ST3Gal UI) from rat liver transfers neuraminic acid from CMP-neuraminic acid to the 3-OH function of terminal Gal residues in Galßl- 4GlcNAc or Galßl → θGlcNAc sequences, such as This is also shown in FIG. 3, while α2.6-SialT (α2.6-SialT, EC 2.4.99.1) transfers neuraminic acid to the 6-OH function of terminal Gal residues in Galßl-4GlcNAc (cf. FIG. 4 ) (Harduin-Lepers, A., Recchi, M.-A., Delannoy, P., Glycobiology 1995, 5, 741; Tsuji, S., Datta, AK, Paulson, J. C, Glycobiology 1996, 6, v ).

In view of these diverse and important functions of glycosyltransferases, various test methods for the detection and activity determination of glycosyltransferases have been developed. Different types of sugar nucleotides are implemented with different types of acceptors. The sugar nucleotide is labeled with ³ H, ¹⁴ C or a fluorescent group.After transfer by the corresponding glycosyltransferase, the now labeled product can be measured and detected in a variety of ways, such as by measuring the radioactivity according to C18 Sep-Pak, HPLC, thin ^¬ layer chromatography, extraction using organic solvents or exclusion chromatography (Palcic, MM, Heerze, LD, Pierce, M., Hindsgaul, O., Glycoconj. J. 1988, 5, 49; Salvucci, ME, Crafts-Brandner, SJ, Anal. Biochem. 1991, 194, 365; Sadler, JE, Beyer, T. A., Oppenheimer, CL, Paulson, J. C, Prieels, J.-P., Rearick, J. I, Hill, RL, Methods Enzymol. 1982, 83, 458; Gu, XB, Gu, TJ, Yu, RK, Analt Biochem. 1990, 185, 151; Dakour, J., Zopf, D., Lundblad, A., Analt. Biochem. 1992, 204, 210; Spicer, AP, Olson, JS, McDonald, JA, J. Biol. Chem. 1997, 272, 8957; Shukla, GS, Radin, NS, Arch. Biochem. Biophys. 1990, 283, 372; White, AR, Xin, Y., Pezeshk, V., Biochem. J. 1993, 294, 231; Nakamura, M., Tsunoda, A., Saito, M., Analt. Biochem. 1991, 198, 154). Other assays use labeled sugar acceptors (Higuchi, T., Tamura, S., Takagaki, K., Nakamura, T., Morikawa, A., Tanaka, K., Tanaka, A., Saito, Y., Endo, M., J. Biochem. And Biophys Methods. 1994, 29, 135; Lee, KB, Desai, UR, Palcic, MM, Hindsgaul, O., Linhardt, RJ, Analt. Biochem. 1992, 205, 108; Liu, AH, Liu, F., Li, Z., Gu, JX, Chen, HL, Cell Biol. Int. 1998, 22, 545) or no markings of the acceptors or donors. The glycosyltransferase activity is determined by ELISA-based methods, coupled enzyme assays, microtiter plate-based fluorescence assays, mass spectroscopy or by means of HPLC (Crawley, S.C, Hindsgaul, O., Alton, G., Pierce, M. , Palcic, MM, Analt. Biochem. 1990, 185, 112; Gosselin, S., Alhussaini, M., Streiff, MB, Takabayashi, K., Palcic, MM, Analt. Biochem. 1994, 220, 92; Shedletzky, E., Unger, C, Delmer, DP, Analt. Biochem. 1997, 249, 88; Kodama, H., Kajihara, Y., Endo, T., Hashimoto, H., Tetrahedron Lett. 1993, 34, 6419) ,

Disadvantages of the test methods available hitherto for the investigation of the enzymatic glycosyltransferase activity are their limited sensitivity and specificity as well as their inadequate usability for screening a large number of samples (High Throughput System = HTS) as is required for a diagnostic application, for example for the diagnosis of tumors ( Preventive care, intraoperative rapid diagnosis, follow-up and follow-up) would be desirable.

The object of the invention is to provide a method for the detection or screening of the enzymatic glycosyltransferase activity which overcomes the above-mentioned disadvantages of the methods according to the prior art and can also be used in particular in HTS and in the diagnosis of diseases.

According to the invention, the object is achieved in a first aspect by a method for detecting the enzymatic glycosyltransferase activity in a sample, wherein the sample is reacted together with a glycosyl donor and a carrier, in particular a cellulose membrane, immobilized glycosyl acceptor, and

in the presence of an enzymatic glycosyltransferase activity, a transferase product is detected.

In one embodiment it is provided that the glycosyl donor is present in excess. In a preferred embodiment it is provided that the excess corresponds to at least twice the Km value of the glycosyltransferase to be detected.

In one embodiment it is provided that the glycosyl acceptor is selected from the group comprising monosaccharides, oligosaccharides, polysaccharides and glycoconjugates. In a preferred embodiment it is provided that the glycosyl acceptor is part of an oligosaccharide.

In a further preferred embodiment it is provided that the glycoconjugate is selected from the group comprising glycoproteins, glycopeptides and glycolipids.

In a particularly preferred embodiment it is provided that the glycoconjugate is a lactosamine derivative, in particular an N-acetyllactosamine derivative.

In one embodiment it is provided that the glycosyl acceptor is immobilized on the membrane directly or via a linker system.

In a preferred embodiment it is provided that the linker system is selected from the group comprising light cleavable linkers (photolinkers) and ester linkers. Carboxylic ester linkers are particularly preferred and glycine ester linkers are very particularly preferred.

In a further embodiment it is provided that the glycosyl donor is a nucleotide sugar. In a preferred embodiment it is provided that the nucleotide sugar is selected from the group consisting of UDP-Gal, UDP-GalNAc, UDP-Glc, UDP-GlcNAc, UDP-Xyl, UDP-GluA,, GDP-Man, GDP-Fuc and CMP-NeuAc includes. The nucleotide sugar CMP-NeuAc is particularly preferred.

In one embodiment it is provided that the glycosyl donor carries a label or a chemoselective function, which preferably enables later labeling.

In a preferred embodiment it is provided that the label is selected from the group comprising fluorescence labels, radioactivity labels, enzyme activity labels and chemiluminescence labels.

In a further embodiment it is provided that the glycosyltransferase is selected from the group comprising galactosyltransferases, fucosyltransferases, sialyltransferases, glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, glucuronyltransferases and glucosamine transferases.

In a preferred embodiment it is provided that the galactosyltransferase is a beta-1,3- or beta-1,4-galactosyltransferase.

In an alternative preferred embodiment it is provided that the fucosyltransferase is selected from the group comprising alpha-1, 3/4 fucosyltransferases and alpha-1,2-fucosyltransferases.

In a further alternative preferred embodiment it is provided that the sialyltransferase is selected from the group comprising alpha-2,3-sialyltransferases and alρha-2,6-sialyltransferases.

In one embodiment it is provided that the sample is selected from the group comprising human, animal, plant and microbial samples. In a preferred embodiment it is provided that the sample is selected from the group comprising tissue and body fluids.

In a very particularly preferred embodiment it is provided that the tissue is tissue containing Golgi apparatus.

In a further embodiment it is provided that the sample is selected from the group comprising cell lysates, tissue lysates, organ lysates, extracts, crude extracts, purified preparations and unpurified preparations.

In a further preferred embodiment it is provided that the body fluid is selected from the group comprising breast milk, urine, sputum, saliva, serum and lymph fluid.

In a further aspect of the invention, the object is achieved by a method for parallel detection of the enzymatic activity of glycosyltransferase (s) in a large number of samples, characterized in that the method according to the invention is carried out in parallel for each sample.

In a further aspect of the invention, the object is achieved by a method for determining the glycosyltransferase pattern of a sample, it being provided that

an aliquot of a sample is reacted together with a first combination of a glycosyl donor and a glycosyl acceptor immobilized on a support, in particular a cellulose membrane, and it is determined whether a transferase product is formed, and

another aliquot of a sample is reacted together with a second combination of a glycosyl donor and a glycosyl acceptor immobilized on a support, in particular a cellulose membrane, and it is determined whether a transferase product is formed. In one embodiment it is provided that the method further comprises the step of determining the transferase product, the determination of the transferase product preferably comprising its biological and / or chemical and / or physical characterization.

In a still further aspect of the invention, the object is achieved by a method for determining the substrate specificity of a glycosyltransferase, wherein

the glycosyltransferase is provided in a sample,

an aliquot of the sample is reacted together with a first combination of a glycosyl donor and a glycosyl acceptor immobilized on a support, in particular a cellulose membrane, and it is determined whether a transferase product is formed, and

another aliquot of the sample is reacted together with a second combination of a glycosyl donor and a glycosyl acceptor immobilized on a support, in particular a cellulose membrane, and it is determined whether a transferase product is formed.

In one embodiment it is provided that the method further comprises the step of determining the transferase product, the determination of the transferase product preferably comprising its biological and / or chemical and / or physical characterization.

In one aspect of the invention, the object is achieved by using one of the methods according to the invention for diagnosing and / or monitoring the course of diseases which are associated with an altered glycosyltransferase activity.

In one embodiment it is provided that the disease is selected from the group comprising cirrhosis of the liver, immune deficiency diseases, lysosomal storage diseases and tumor diseases. In a preferred embodiment it is provided that the tumor disease is selected from the group comprising human and animal carcinomas.

In a particularly preferred embodiment it is provided that the carcinoma is selected from the group comprising melanomas, breast carcinomas, colon carcinomas, choriocarcinomas, hematoma carcinomas and bladder cancer.

In one embodiment of the method according to the invention it is provided that the individual reaction steps are carried out in parallel, preferably highly parallel.

One application of this embodiment is a high throughput system in which the detection of the enzymatic glycosyltransferase activity, the determination of the glycosyltransferase pattern and / or the determination of the substrate specificity of a glycosyltransferase is carried out simultaneously for a large number of reaction batches.

In a further aspect, the object is achieved by a method for identifying a compound influencing the expression of a glycosyltransferase, comprising the following steps:

Providing a candidate connection,

Testing the candidate compound for its ability to affect the expression of a glycosyltransferase in an expression system, wherein

the expression of the glycosyltransferase is determined in that a glycosyl acceptor immobilized on a support, in particular a cellulose membrane, and a glycosyl donor are present in the expression system,

and the presence or absence of a transferase product is detected.

In one embodiment it is provided that the compound inhibits the expression of the glycosyltransferase. In a further embodiment it is provided that the expression is the transcription.

In an alternative embodiment it is provided that the expression is translation.

In a further aspect of the invention, the object is achieved by a method for identifying a compound influencing the activity of a glycosyltransferase, comprising the following steps:

Providing a candidate connection,

Testing the candidate compound for its ability to affect the activity of a glycosyltransferase in a reaction, where

the glycosyltransferase activity is determined in that a glycosyl acceptor immobilized on a support, in particular a cellulose membrane, and a glycosyl donor are present in the reaction mixture,

and the presence or absence of a transferase product is detected.

In one embodiment it is provided that the compound inhibits the activity of the glycosyltransferase.

In a further embodiment it is provided that the substeps of the method for identifying a compound influencing the expression of a glycosyltransferase and / or the substeps of the method for identifying a compound influencing the activity of a glycosyltransferase are carried out in parallel with at least two different candidate compounds.

In a still further embodiment of the method according to the invention it is provided that the candidate compound is contained in a library of compounds, preferably a library of low molecular weight compounds. In one aspect of the invention, the object is achieved by a kit according to the invention for the detection of an enzymatic glycosyltransferase activity comprising at least one glycosyl acceptor immobilized on a support, preferably a cellulose membrane.

In one embodiment it is provided that the kit comprises a glycosyl donor.

In a further embodiment it is provided that the kit comprises at least one further component which is selected from the group comprising buffers, glycosyl donor (s), glycosyl acceptor (s) and glycosyl transferase (s).

The present invention is based on the surprising finding that immobilizing a glycosyl acceptor on a support, in particular a membrane and preferably a cellulose membrane, provides a highly sensitive method for studying the activity of glycosyltransferases in the broadest sense. The high sensitivity and selectivity of the method allows the determination of the glycosyltransferase activities for all cases in which the sample material is only available to a limited extent, such as in crude extracts. At the same time, one advantage of the method according to the invention is that it is precisely because of this high sensitivity that the method is suitable for high-parallel analysis methods (high throughput screening). The investigation of the enzymatic activity of glycosyltransferases can be divided into different aspects. One aspect of the method relates to the detection of one or the enzymatic glycosyltransferase activities in a sample, a second aspect to the determination of the glycosyltransferase activity pattern of a sample and a further aspect to the determination of the substrate specificity of a glycosyltransferase. All of these processes share the above-mentioned use of the immobilized glycosyl acceptor and cause the surprising advantages of the system.

The term detection of glycosyltransferase activity in a sample generally describes detection here and includes both qualitative and quantitative detection of glycosyltransferase activity. The type of glycosyltransferase that can be determined in the chosen approach depends on the one hand on the glycosyl donor or immobilized glycosyl acceptor used and on the other hand on the chosen reaction methods. conditions that are chosen in the verification procedure. It is within the knowledge of the person skilled in the art in this field to select the possible glycosyl donors, the possible glycosyl acceptors and the specific reaction conditions for the glycosyltransferase activity to be detected in each case and thus to ensure the specificity of the detection of a specific glycosyltransferase activity.

When determining the glycosyltransferase activity pattern of a sample, it is assumed that one or more glycosyltransferases are contained in the sample. By using different combinations of glycosyl donor and glycosyl acceptor, the enzymatic activities of different glycosyl transferases can be determined. It is also within the scope of the present invention to use different combinations of glycosyl donor (s) and glycosyl acceptor (s) with the aim, even without knowledge of the specific combinations, for the individual glycosyltransferase activity (s) to be detected. to generate a pattern due to the combinations consisting of the parameters glycosyl donor and glycosyl acceptor. Such a pattern can be quite specific for a certain sample and the recognition of a certain state, e.g. B. a disease state, or to identify the type of a specific sample, without the actual specificities of the glycosyltransferase contained in the sample necessarily having to be known.

When determining the substrate specificity of a glycosyltransferase, it is assumed that one glycosyltransferase or several glycosyltransferases are present in a sample and that the substrate specificity of at least one of the glycosyltransferases is determined. The substrate specificity can be determined both with regard to the glycosyl donor and the glycosyl acceptor. In both cases, a number of possible glycosyl donors or glycosyl acceptors will be used in a corresponding approach using the respectively immobilized glycosyl acceptor. From the combination of different glycosyl donors and glycosyl acceptors, the substrate specificity of the glycosyl transferase to be investigated can then be determined. H. be derived.

In the method according to the invention described herein, the glycosyltransferase is typically contained in a sample or the sample is one in which a glycosyltransferase may be present. Typically, a predetermined amount of sample is added labeled glycosyl donor and the glycosyl acceptor immobilized on the preferably used cellulose membrane. The appropriate reaction conditions are selected with regard to the glycosyl transferase to be investigated and the glycosyl donor present. It is entirely within the scope of the invention that the sample already contains the glycosyl donor and / or the non-immobilized glycosyl acceptor. The reaction conditions include reaction time, pH and temperature, which allow an effective reaction of one or the glycosyltransferase with the labeled glycosyl donor and the glycosyl acceptor. The selection of the corresponding reaction conditions is within the knowledge of the person skilled in the art in this field and is particularly known or easy to determine in the event that a specific glycosyltransferase is to be determined in a sample. The usual incubation times are 5 minutes to 24 hours. The pH and the temperature can be in a wide range, not least depending on the glycosyltransferase to be detected. Typical pH values are pH 5 to 8, the typical temperatures are 20 ° C to 45 ° C. The amount of glycosyl donor and immobilized glycosyl acceptor used depends on the glycosyl transferase to be investigated. The concentration of the glycosyl donor should preferably be at least twice the km of the glycosyl transferase to be investigated, typically 0.1 to 10 mM.

The reaction preferably takes place in a buffer system. Systems known in the literature, such as MES, MOPS, sodium cacodylate or Tris, are used as buffers. The buffer serves to maintain an effective pH value for the reaction and, if necessary, an effective ionic strength. Divalent ions such as e.g. B. Ca ²⁺ , Mg ²⁺ or Mn ²⁺ , or detergents can be added as required. Such buffer systems or cations and detergents can constitute further components of the kit according to the invention.

Unreacted reagents, such as excess glycosyl donor, can be removed after the reaction by simply washing the cellulose membrane with buffer solution, such as PBS. The content of the glycosyltransferase in the sample can be determined from the amount of transferase product obtained by known methods.

The transferase product is typically the glycosyl acceptor modified as a result of the glycosyl transferase activity, which has a group or part of the molecule derived from the glycosyl donor. Basically, however, it is also within the scope of the present Invention that instead of the transferase product, the change in the glycosyl donor that occurs as a result of the glycosyl transferase is also measured, ie the glycosyl transferase activity from the difference between the glycosyl donor used before the reaction and the amount of the glycosyl donor still present after the reaction or the amount of the resultant by the the glycosyltransferase-transferred residue or molecular part of reduced glycosyl donor.

Typically, however, the glycosyl donor is marked and in particular that part which is transferred from the glycosyl donor by the glycosyl transferase, provided that it is present in the respective sample in active, in particular enzymatically active form, to the immobilized glycosyl acceptor. Measurements are based on the determined specific activity of the labeled glycosyl donor, for example UDP-Gal, and thus directly provide the amount of glycosyl residue transferred in the reaction. To maintain the specific activity of the reaction, a corresponding standard curve is created. Under the chosen reaction conditions, the transfer generally depends linearly on time, as shown in FIG. 6. From this relationship, the specific activity of the glycosyltransferase reaction can be calculated.

"Transferase product" can thus be understood in the broadest sense to mean a glycosyl donor, of which a group or a part of a molecule has been transferred to a glycosyl acceptor as a result of the glycosyl transferase activity. Furthermore, the term “glycosyl transferase” is understood here to mean any enzymatic activity which leads to a glycosyl transfer. The term "glycosyltransferase" is used synonymously with the term "glycosyltransferase activity". It is therefore within the scope of the present invention that a glycosyltransferase is, for example, also a protein which, in addition to the glycosyltransferase activity, also has further enzymatic activities.

The method chosen for determining the labeled transferase product depends on the type of labeling of the glycosyl donor or on the residue transferred from the glycosyl donor to the glycosyl acceptor as a result of the glycosyltransferase activity. If the label is a fluorescent label, the glycosyltransferase activity can be determined directly by measuring the fluorescence intensity. With radioactive labeling, for example by ³ H, ¹⁴ C or ³² P, the glycosyltransferase activity can be determined accordingly by measuring the radioactivity. Suitable measuring methods can be carried out by scintillation counters or by autoradiography. For example, autoradiography could which should be indicated if the method for the detection of one or the enzymatic glycosyltransferase activity is one which is carried out on a biochip in which a multiplicity of glycosyl acceptors are arranged on a common carrier and the reaction parameters are distinct Locations of the biochip differ with the consequence that different transferase products can arise.

As provided in one embodiment, cleavable linker systems can be used between the glycosyl acceptor and the support, in particular the cellulose membrane. As a result of the cleavage, the transferase products can be cleaved from the membrane and then detected by conventional analysis techniques such as mass spectroscopy, NMR, DC, HPLC etc. The detection methods used in the context of the methods according to the invention are known to those skilled in the art, as stated above. In addition to the detection methods mentioned above, it is also possible to detect a transferase activity or a transferase product in the form of the immobilized glycosyl acceptor modified as a result of the glycosyltransferase activity by means of plasmon resonance by changing the mass of the immobilized glycosyl acceptor, since a minimal mass change of approximately 180 Da is required for plasmon resonance this technique can therefore be used to detect a transfer of monosaccharides by the glycosyltransferase to be detected.

The glycosyl donors can also carry one or more reactive functionalities (optionally provided with protective groups) which, after the reactive functionality has been transferred to the immobilized glycosyl acceptor (optionally after the protective group has been split off) together with the group or molecular part of the glycosyl donor transferred by the glycosyl transferase, initially reacted chemoselectively with a functionalized label and then analyzed. A number of reactions can be used for this purpose, which are known to those skilled in the art as such (Lemieux, GA & Bertozzi, CR, 1998, Chemoselective ligation reactions with proteins, oligosaccharides and cells, TIBTECH, 16, 506-513). Examples of suitable reactions are the formation of thiazolidines from aldehydes and 1,2-aminothiols, of oxazolidines from aldehydes / ketones and 1,2-amino alcohols, of hnidazoles from aldehydes / ketones and 1,2-diamines, of thiazoles from thioamides and alpha-halo-ketones, from aminothiazoles from amino-oxy compounds and alpha-isothiocyanato-ketones, from oximes from amino-oxy compounds and aldehydes, from oxi Men from amino-oxy compounds and ketones, from hydrazone from hydrazines and aldehydes, from hydrazone from hydrazides (see. Fig. 7).

Immobilization of the substrates used, i. H. of the glycosyl acceptors occurs by forming a covalent bond of the saccharide or glycoconjugate with the surface of the support, in particular the cellulose surface. The substrates can be directly, for. B. by reaction of an aldehyde function of the immobilizing, reducing saccharide with an amino-functionalized cellulose surface, by reductive amination or via suitable spacers (English: spacer) / linker, which enable later cleavage, to the support, in particular to the cellulose membrane. Systems which are both linkable and cleavable under mild conditions are particularly suitable as linkers. H. while maintaining the glycosidic bonds, such as, for example, photolabile linkers (T. Ast, N. Heine, L. Germeroth, J. Schneider-Mergener, H. Wenschuh, Tetrahedron Lett. 1999, 40, 4317) or ester linkers (R. Frank, Tetrahedron 1992, 48, 9217). An overview of common linker systems that can be used and used for the present invention is given, for example, in BA Bunin, The Combinatorial Index, Academic Press, 1998, and in D. Obrecht, JM ViUalgordo, Combinatorial and parallel synthesis of small-molecular-weight compound libraries , Elsevier, 1998.

As a result of the immobilization of the immobilized glycosyl acceptor, the methods according to the invention, in particular the sub-step relating to the detection of a transferase product and thus the detection of the presence or quantification of the glycosyltransferase, can comprise a step in which the transferase product or a part thereof from the carrier or the cellulose membrane is split off.

A wide range of immobilized glycosyl acceptors can be used in the process according to the invention described herein. Both monosaccharides and oligosaccharides and polysaccharides can serve as immobilized glycosyl acceptors. Glycoconjugates such as glycopeptides, glycoproteins and glycolipids are also suitable. Examples of polysaccharides which can be used in the context of the present invention as immobilized glycosyl acceptors are starch, glycogen and chitin. Examples of possible glycosyl acceptors that can be used as substrates for glycosyltransferases in the context of the invention disclosed herein are given in Table 1 below.

Table 1: Examples of glycosyl acceptors that act as substrates for glycosyltransferases (B. Ernst, G. W. Hart, P. Sinay, Carbohydrates in Chemistry and Biology 2000, Wiley-VCH, 689-624)

In the methods according to the invention, it is particularly preferred to detect the glycosyltransferase activity in a sample, the sample being breast milk and the glycosyltransferase to be detected being a fucosyltransferase. Another particularly preferred embodiment of the method according to the invention is that in which the glycosyl donor is CMP-NeuAc, the neuraminic acid being provided with a fluorescent label.

In the method according to the invention for determining the glycosyltransferase activity pattern, it can be provided that the aliquot of a sample, which together with a first combination of a glycosyl donor and a glycosyl alkoxylated on a carrier, zeptor is implemented, continues to be used and so far represents another aliquot of a sample. It is also within the scope of the present invention that the sample is processed further between the different steps, for example by specifically removing or inhibiting compounds or enzymatic activities contained in the sample or in the reaction mixture, such as the glycosyltransferase investigated in the context of the use of the first combination , The same applies analogously when determining the substrate specificity of a glycosyltransferase.

The methods according to the invention can be used in particular in the field of diagnostics, follow-up or prognostics. Typically, it is used in diseases that are associated with a change in glycosyltransferase activity. This use is based on the observation that a number of disease states are associated with altered glycosyltransferase activities. It has been observed, for example, that unusual carbohydrate structures (tumor-associated antigens) are found in a large number of cancers (T. Feizi, Nature 1985, 314, 53-57; JW Arends, FT Bosmann, J. Hilgers, Biochem. Biophys. Acta 1985, 780 , 1-19; S. Hakamori, Y. Zhang, Chem. Biol. 1991, 4, 91; SJ Danisefsky, JR Allen, Angew. Chem. Int. Ed. 2000, 39, 836-863) on the cell surface of the tumor cells are detected, which do not occur in normal tissue or only occur in significantly smaller amounts (S. Hakamori, Adv. Cancer Res. 1989, 52, 257). These are mostly glycolipids of the lacto or neolacto type. In addition to the accumulation of normal blood group antigens such as Le, Le or Le ^{x, there} are also more complex structures that are altered by additional fucose or neuraminic acid residues and by dimerization or trimerization. The unusual glycosylation patterns are often the direct consequence of changed enzyme activities of the corresponding glycosyltransferases (S. Narasimhan, H. Schachter, S. Rajalakshimi, J. Biol. Chem. 1988, 263, 1273-1281; K. Yamashita, T. Okhura, Y. Tashibana, S. Takasaki, A. Kobata, J. Biol. Chem. 1984, 259, 10834-10840; M. Pierce, J. Arango, J. Biol. Chem. 1986, 261, 10772-10777).

As a result of the universal applicability of the methods according to the invention, the various structures which are based on the expression or activity patterns of glycosyltransferases on which the various disease states are based can thus be assigned to these. Table 2 shows an overview of tumor-associated glycosphingolipids with the monoclonal antibodies used for their detection, which can be used as glycosyl acceptors in the process according to the invention.

Tab. 2 References to Table 2:

1 W. W. Young, H. S. Johnson, Y. Tamura, K.-A. Karlsson, G. Larson, J.M.R. Parker, D.P. Khare, U. Spohr, D.A. Baker, O. Hindsgaul, R.U. Lemieux, J.Biol. Chem. 1983, 258, 4890-4894.

2 M. Blaszczyk, G C. Hansson, K.-A. Karlsson, G. Larson, N. Strömberg, J. Thurin, M. Herlyn, Z. Steplewski, H. Koprowski, Arch. Biochem. Biophys., 1984, 233, 161-168.

3 J.L. Magnani, B. Nilsson, M. Brockhaus, D. Zopf, Z. Steplewski, H. Koprowski, V. Ginsburg, J. Biol. Chem. 1982, 257, 14365-14369.

4 H. Koprowski, Z. Steplewski, K. Mitchell, M. Herlyn, D. Herlyn, P. Fuhrer, Somat. Cell Genet. 1979, 5, 957-972.

5 D. Chia, P.I. Terasaki, N. Suyama, J. Galton, M. Hirota, D. Katz, Cancer Res. 1985, 45, 435-437.

6 M. R. Stroud, S. B. Levery, M. E. K. Salyan, C. E. Roberts, S. Hakamori, Eur. J. Biochem. 1992, 203, 577-586.

7 H. Ito, K. Tashiro, M.R. Stroud, T.F. 0ntntoft, P. Meldgaard, A.K. Singhai, S. Hakamori, Cancer Res. 1992, 52, 3739-3745.

8 K. Abe, J.M. McKibbin, S. Hakamori, J. Biol. Chem. 1983, 258, 11793-11797.

9 A. Brown, T. Feizi, H.C. Gooi, M.J. Embleton, J.K. Picard, R.W. Baldwin, Biosci. Reports 1983, 3, 163-170.

10 K.O. Lloyd, G. Larson, N. Strömberg, J. Thurin, K.-A. Karlsson, Immunogenetics 1983, 17, 537-541.

11 T. Kaizu, S. B. Levery, E. D. Nudelman, R. E. Stenkamp, S. Hakamori, J. Biol. Chem. 1986, 261, 11254-11258.

12 E. D. Nudelman, S. B. Levery, T. Kaizu, S. Hakamori, J. Biol. Chem. 1986, 261, 11247-11253.

13 K. Fukushima, M. Hirota, P.I. Terasaki, A. Wakisaka, H. Togashi, D. Chia, N. Suyama, Y. Fukushi, E. D. Nudelman, S. Hakamori, Cancer Res. 1984, 44, 5279-5285.

14 Y. Fukushi, E. D. Nudelman, S. B. Levery, H. Rauvala, S. Hakamori, J. Biol. Chem. 1984, 259, 10511-10517.

It was also observed that tumor cells often have both more complex and terminally modified (fucosylated, sialylated) N and O glycan structures. Some examples of glycan structures found on tumor cells and the main responsible glycosyltransferases can be found in Table 3:

Tab. 3

References to Table 3:

15 a) M. Pierce, J. Arango, J. Biol. Chem. 1986, 261, 10772-10777. b) J. W. Dennis, S. Laferte, C. Waghorne, M. L. Breitman, R. S. Kerbel, Science 1987, 236, 582-585. c) K. Yamashita, T. Ohkura, Y. Tachibana, S. Takasaki, A. Kobata, J. Biol. Chem. 1984, 259, 10834-10840. d) J.W. Dennis, K. Kosh, D.-M. Bryce, M.L. Breitman, Oncogene 1989, 4, 853-860. e) E. Miyoshi, A. Nishikawa, Y. Ihara, J. Gu, T. Sugiyama, N. Hayashi, H. Fusamoto, T. Kamada, N. Taniguchi, Cancer Res. 1993, 53, 3899-3902.

16 A. Kobata, J. Cell Biochem. 1988, 37, 79-90.

17 a) S. Narashima, H. Schachter, S. Rajalakshmi, J. Cell Biol. 1988, 263, 1273-1281. b) 18e)

18 a) S. H. Itzkowitz, M. Yan, Y. Fukushi, A. Palekar, P. C. Phelphs, A. M. Shamsuddin, B. F.

Trump, S. hakamori, Y. S. Kim, Cancer Res. 1986, 46, 2627-2632. b) S.-I. Hakomori, Adv. Cancer Res. 1989, 52, 257-331. c) K. D. Lloyd, Am. J. Clin. Pathol. 1987, 87, 129-139.

19 a) S. Yousefi, E. Higgins, Z. Doaling, O. Hindsgaul, A. Pollex-Kruger, J. W. Dennis, J.

Biol. Chem. 1991, 266, 1772-1783. b) K. Shimodaira, J. Nakayama, N. Nakamura, O. Hasebe, T. Katsuyama, M. Fukuda, Cancer. Res. 1991, 51, 5201-5206.

As a result of this and the substrate specificities or glycosyltransferase activity patterns that can be determined in principle using the methods according to the invention, an effective diagnosis, follow-up control and prognostic system can be set up. In this context, the term follow-up control means in particular that the corresponding glycosyltransferase activity, the glycosyltransferase pattern or the substrate specificity of the glycosyltransferase is determined before the start of therapy, and samples are again taken from the treated individual in the course of a treatment and examined for precisely these parameters. Is there a correlation between the corresponding parameters and the course of the disease? If, for example, the activity of certain glycosyltransferases goes hand in hand with increasing deterioration of the disease or increased tumor growth, appropriate conclusions can be drawn from the determined parameters as a function of the course of treatment.

In principle, the same also applies to the prognostic application of such systems, with prognosis being understood here in particular as meaning statements regarding the probability of recovery of a patient, whether under the influence of a treatment or without. For this purpose, in addition to the correlation described above, a further correlation with the respective parameters is required in such a way that a statement from the course of the parameters or their change or non-change over time with the probability of recovery, stabilization of a condition or updating of a condition or deterioration of the condition of an individual.

In a further aspect, the invention relates to methods for identifying a compound which influences the expression of a glycosyltransferase. The term expression is to be understood here in particular in such a way that it can be both transcription and translation. As a result of the use of an immobilized glycosyl acceptor in combination with a glycosyl donor and a glycosyl transferase, this method can also be designed particularly effectively in terms of use in a high throuput system (HTS). The compounds influencing the expression are preferably inhibitors. The inhibitors can reduce or completely prevent transcription or translation of the glycosyltransferase. With the increased sensitivity of the method due to the specific procedure, in particular the use of the immobilized glycosyl acceptor on a cellulose membrane, even minor changes in the expressed glycosyltransferase can thus be detected and a differentiation of the various inhibitors can thus be carried out. The same applies in principle to those compounds which increase the expression of a glycosyltransferase. The compound influencing expression is also referred to herein as a candidate compound, especially if it is used for the first time in such a test system and it is not known to this extent whether and if so to what extent it influences the expression of a glycosyltransferase. As with the other methods of the invention disclosed herein, the glycosyltransferase can be virtually any glycosyltransferase. but preferably such as is used in connection with the other methods of the invention described herein. The same applies to the glycosyl donors or glycosyl acceptors and the respective process parameters.

In the methods according to the invention for identifying a compound influencing expression, said compound can influence either transcription or translation, i.e. inhibit or stimulate, said compound preferably being an inhibitor.

After the features of the method according to the invention for the detection of a glycosyltransferase are contained in the above methods and for the assessment of the question whether, and if so, to what extent a candidate compound actually inhibits or stimulates expression, ultimately the glycosyltransferase activity in the reaction mixture Further steps are typically required to determine at which level of expression the candidate compound actually intervenes in the reaction. This can be done, for example, by examining whether, and if so to what extent, the mRNA coding for the particular glycosyltransferase is present in the expression system. If the amount of mRNA is reduced under the influence of the candidate compound with reduced glycosyltransferase activity, it can be assumed that the inhibitory effect of the candidate compound takes place at the level of the transcription. The methods for determining the amount of mRNA are known to those skilled in the art. The same considerations apply mutatis mutandis in the event that the candidate compound has a stimulating effect.

Conversely, if the glycosyltransferase activity is reduced under the influence of a candidate compound and the amount of mRNA remains unchanged, it can be assumed that the inhibitory effect of the candidate compound occurs at the translation level. Under such conditions, the amount of translated protein which has the glycosyltransferase would generally also be reduced. The methods for determining the amount of translated protein containing the glycosyltransferase are known to those skilled in the art. As an example, reference is made to antibodies against said protein. The same considerations apply mutatis mutandis in the event that the candidate compound has a stimulating effect. “Expression system” is also to be understood here as meaning a cellular system, a cell-free system or a cell extract. However, the term “expression system” can also be understood to mean a system in which only the aspect of translation or the aspect of transcription is examined. In any event, the phenotype is, in a sense, ultimately based on the presence or absence of a glycosyltransferase, which is detected according to the method disclosed herein for the detection of glycosyltransferases.

The method claimed in a further aspect of the present invention for identifying an activity, i. H. the compound influencing the enzymatic glycosyltransferase activity. Here again, it is preferably again an inhibitor which inhibits the glycosyltransferase activity, i.e. H. reduced or completely prevented. As a rule, the candidate compound is tested in a reaction mixture which, in addition to the glycosyltransferase, typically also comprises a glycosyl donor and an immobilized glycosyl acceptor, which is preferably immobilized on a cellulose membrane. As with all of the methods according to the invention disclosed herein, the detection of one or the enzymatic glycosyltransferase activity takes place via the detection (or non-detection) of the presence or absence of a transferase product, which gives direct conclusions about the presence or absence or the specific glycosyltransferase activity allows, since a transferase product only arises under the influence of a glycosyltransferase, as can easily be determined by appropriate control approaches (negative controls and positive controls), as is familiar to the person skilled in the art in this field.

All of the methods according to the invention disclosed herein have in common that they are suitable for the needs of high-through-put systems due to the high sensitivity, which not least allows a reduced sample volume. Furthermore, the immobilization of the glycosyl acceptor opens up the possibility of detecting a site-specific reaction, which is particularly advantageous in the detection of the transferase product, since it avoids handling liquid quantities, which are usually a limitation of high throughput systems. Finally, in a further aspect, the object on which the invention is based is achieved by a kit. This kit can generally be used for or in the context of all of the methods and uses disclosed herein. The kit is also based on the knowledge on which the invention is based, that of highly sensitive detection of glycosyltransferase activities using an immobilized glycosyl acceptor. It is also within the scope of the present invention that, instead of the glycosyl acceptor already immobilized on the support, in particular a cellulose membrane, such a kit also comprises a membrane and a glycosyl acceptor, and optionally reagents for immobilizing the glycosyl acceptor on or on (both terms are synonymous here used) the carrier included in the kit. To this extent, the term in connection with the kit of a glycosyl acceptor immobilized on or on a carrier is also understood to mean that it is only - typically - produced from the individual components immediately before use.

In addition to these components, the kit according to the invention can comprise further components and constituents, as are provided in prior art kits. Such components may include, among other things, positive and negative controls, one or more glycosyl transferases, one or more glycosyl donors, one or more glycosyl acceptors, and suitable buffers.

The invention will now be explained below with reference to the figures and examples, from which further features, advantages and embodiments of the invention result. It shows

1 shows the transfer of galactose catalyzed by an α1, 3 galactosyltransferase;

2 shows the transfer of fucose catalyzed by an α 1,3 / 4 fucosyltransferase;

3 shows the transfer of neuraminic acid catalyzed by an α2,3- (N) -sialyltransferase;

4 shows the transfer of neuraminic acid catalyzed by an α2,6- (N) -sialyltransferase;

5 shows the immobilization of LacNAc-O- (CH2) ₈ COOH on an amino-functionalized cellulose membrane; 6 shows the time-dependent enzymatic transfer in the presence of α1, 3-galactosyltransferase;

7 shows an overview of different chemoselective reactions;

8 is a graphical representation of a non-linear regression calculation for transferred galactose;

9 shows the time-dependent enzymatic transfer in the presence of α1, 3-fucosyltransferase;

10 shows the fluorescence result with the α2,3- (N) -sialyltransferase; and

11 shows the result of an autoradiography of the 1,3-galactosyltransferase.

The different figures show the following in detail:

Figure 1: The enzyme αl, 3-galactosyltransferase catalyzes the transfer of Gal from UDP-Gal to the 3-hydroxyl function of the Gal residue from Galßl- »4GlcNAc-R to the compound Galαl → 3Galß l-» 4GlcNAc-R.

Figure 2: The enzyme α1, 3/4 fucosyltransferase catalyzes the transfer of Fuc from GDP-Fuc to Gal residues in structures such as Galßl-> 4GlcNAc-R (Type II) and Galßl → 3GlcNAc-R (Type I) to Le ^x and Le ^a structures.

Figure 3: The enzyme α2,3- (N) -sialyltransferase catalyzes the transfer of neuraminic acid from CMP-neuraminic acid to the 3-OH function of the Gal residues in Galßl → 4GlcNAc-R (type H) or Galßl → 3GlcNAc-R (type I) containing structures.

Figure 4: The enzyme α2,6- (N) -sialyltransferase catalyzes the transfer of neuraminic acid from CMP-neuraminic acid to the 6-OH function of the Gal residues in structures containing Galßl → GlcNAc-R or Galßl → 3GlcNAc-R. Figure 5: shows the derivatization of an amino-functionalized cellulose membrane (glycine ester, photo linker membrane) with the acceptor LacNAc. a) The amino functions of the cellulose membrane are derivatized with the activated ß-alanine building block 'Fmoc-ß-Ala-OPfp' b) After acetylation of the remaining amino groups with acetic anhydride, c) the Fmoc protective group is cleaved with piperidine and d) the LacNAc building block Pfp ester immobilized. Finally, remaining amino functions are acetylated with acetic anhydride.

a) Fmoc ß-Ala-OPfp, NMP; b) 10% Ac ₂ O in MeOH; c) piperidine in DMF; d) LacNAc-O- (CH ₂ ) ₈ COOH, P OH, DIC, NMP; e) 10% Ac ₂ O in MeOH.

X = Gly for glycine ester membrane, X = Photo linker for photolinker membrane.

Figure 6: Shows the time-dependent enzymatic transfer of UDP-Gal (1.2 mM) in the presence of α1, 3-galactosyltransferase (3.3 μU) to the immobilized acceptor LacNAc (see. Example 1, time-dependent transfer experiment). The amount of galactose transferred increases linearly with time (transfer rate 12 fmol / min / mm ² , maximum transfer 50 pmol / mm ² ).

Figure 7: Shows an overview of various chemoselective reactions according to the prior art: A) aldehydes (R ⁴ = H) or ketones (R ⁴ not H) and amino-oxy compounds react to oximes, B) aldehydes (R ⁴ = H) ) or ketones (R ⁴ not H) and thiosemicarbazides react to thiosemicarbazones, C) aldehydes (R ⁴ = H) or ketones (R ⁴ not H) and hydrazides react to hydrazones, D) aldehydes (R ⁴ = H) or ketones ( R ⁴ not H) and 1,2-aminothiols react to thiazolines (X = S) and 1,2-amino alcohols to oxazolines (X = O) and 1,2-diamines react to imidazolines (X = NH), E ) Thiocarboxylates and α-halocarbonyls react to thioesters, F) thioesters and ß-aminothiols react to ß-mercaptoamides, F) mercaptans and maleimides react to succinimides. One of the reactants can be covalently linked via R ¹ or R ⁴ , R ⁵ or R ⁶ or via a linker to the glycosyl donor. FIG. 8: shows a graphical representation of a non-linear regression calculation for transferred galactose as a function of the UDP-Gal concentration for the 1,3-galactosyltransferase (see Example 1, determination of the donor Kim).

Figure 9: Shows the time-dependent enzymatic transfer of GDP-Fuc (1.0 mM) in the presence of the α1, 3-fucosyltransferase (0.65 mU) to the immobilized acceptor LacNAc (see Example 2, time-dependent transfer experiment). The transfer rate of GDP-Fuc with 656 μU was determined with 0J8 fmol / min / mm ² , and the maximum transfer with 50 pmol / mm ² .

Figure 10: Shows the result of the transfer of fluoresceinyl-neuraminic acid by the α2,3- (N) -sialyltransferase (see Example 6). CMP-5-fluoreseeinyl Νeuramic acid is transferred to the immobilized acceptor LacNAc in the presence of the α2,3- (N) -sialyltransferase. The transfer was confirmed after 2, 4 and 24 h by fluorescence.

A: Control membrane without addition of transferase B LacNAc bound to glycine ester membrane C time-dependent transferase experiment (2h, 4h, 24h)

Figure 11: Shows the autoradiography results of the α1, 3-galactosyltransferase after 6 days of exposure (see Example 7).

ester 1: glycine ester membrane without immobilized LacNAc (negative control) ester 2: LacΝAc glycine ester membrane photolinker 1: photolinker membrane without immobilized LacNAc (negative control) photo linker 2: LacNAc photolinker membrane

The membrane strips were incubated for 24 h with ¹⁴ C-UDP-Gal in the presence of different 1,3-GalT concentrations: a) 132 μU b) 13.2 μU c) 1.32 μU.

A clear transfer can be demonstrated at an α 1,3-GalT concentration of 132 μU both for the LacNAc glycine ester membrane and the LacNAc photolinker membrane. With a 1,3-GalT concentration of 13.2 μU, the transfer can only be Show NAc photolinker membrane (higher loading with LacNAc). The corresponding negative controls (Ester 1, Photolinkerl) show negligible (a) or no (b) signals.

The following abbreviations are used in the following examples:

AP Alkaline Phosphatase

BHK baby hamster kidney

BSA Bovine Serum Albumin, bovine serum albumin

CMP cytidine 5 'monophosphate

DC thin layer chromatogram

DIC N, N-diisopropyl carbodiimide

DMF N, N-dimethylformamide dpm decays per minute

DTT 1,4-dithio-DL-threitol

ELISA enzyme-linked immunosorbent assay

Fmoc 9-fluorenylmethoxycarbonyl

Fmocß-Ala-OPfp N-ß-Fmoc-ß-alanine pentafluorophenyl ester

Fuc L-fucose

FucT, FT fucosyltransferase

Gal D-galactose

GalNAc N-acetyl-D-glucosamine

GalT galactosyltransferase

GDP guanosine 5 'diphosphate

Glc D-glucose

GlcA D-glucuronic acid

GlcNAc N-acetyl-D-glucosamine

Gly glycine hour, hour

HPLC high-performance liquid chromatography

HTS High throuput screening

L liter

LacNAc Galß (l → 4) GlcΝAc

LacNAc-O- (CH ₂ ) ₈ COOH Galß (l → 4) GlcNAcß (l → l) -8-octanoic acid

Le Lewis

Le ^a Galß (l → 3) [Fucα (l → 4)] GlcNAc

Le ^x Galß (l → 4) [Fucα (l-> 3)] GlcNAc

Le ^y Fucα (l- »2) Galß (l- 4) [Fuc (l- → 3)] GlcNAc

M molar mAb monoclonal antibody

Man D-Mannose

MeOH methanol

MES 2-morpholino-ethanesulfonic acid monohydrate mL milliliter mM millimolar

MOPS 3 -morpholino-propanesulfonic acid mU milliunits

Neu5Ac, NeuAc N-acetylneuraminic acid

NMP 1 -methyl-2-pyrrolidone

NMR nuclear magnetic resonance

PBS phosphate buffered saline

PfpOH 2,3,4,5,6-pentafluorohenol

R molecular residue

SialT, ST sialyltransferase

Sialyl-Le ^x , S-Le ^x ΝeuAcα (2 → 3) Galß (l → 4) [Fucα (l → 3)] GlcΝAc

Tris 2-amino-2-hydroxymethyl-1,3-propanediol

U unit

UDP uridine 5 'diphosphate

UV ultraviolet

Xyl D-xylose

The following chemicals, derivatizations of the cellulose membranes and reaction conditions for the cleavage of the compounds from the cellulose membrane were used for the examples described below.

chemicals

Cellulose membranes (filter paper 50) were manufactured by Whatman International Ltd. (Maidstone, England). l-Methyl-2-pyrrolidone (NMP), pentafluorophenol (PfpOH), 1,3-diisopropylcarbodiimide (DIC), and acetic anhydride are from Sigma-Aldrich (Germany). Methanol and NN-dimethylformamide (DMF) come from Merck Eurolab GmbH (Germany). The photolinker 4- [4 (l-Fmoc-aminoethyl) -2-methoxy-5-nitrophenoxyl] - butanoic acid, 1,3-fucosyltransferase (α (l- »3) FT), and α 2,6-sialyltransferase (α (2- »6) ST) were obtained from Calbiochem-abiovabiochem (San Diego, USA). N ^α -Fmoc-ß-Ala-OPfp comes from Bachern Biochemica GmbH (Heidelberg, Germany). CMP-5-fluoresceinyl-ureuric acid, tripotassium salt (CMP-5-fluoro-Νeu5Ac) was obtained from Boehringer-Mannheim (Mannheim, Germany). UDP- [ ¹⁴ C] Gal, GDP- [ ^{, 4} C] Fuc, CMP- [ ¹⁴ C] Neu5Ac are from American Radiolabeled Chemicals, Inc. (St. Louis, USA). UDP-Gal was obtained from Pharma Waldhof GmbH (Germany). GDP-Fuc was a gift from Dr. R. Öhrlein, Ciba-Geigy (Switzerland). CMP-Neu5Ac comes from Juelich Enzyme Products (Germany). Alkaline phosphatase was purchased from Boehringer Mannheim (Mannheim, Germany). Diethylamine, reagent grade comes from Fisher (Fair Lawn, USA). Ecolite Scintillation Cocktail was developed by ICN (Costa Mesa, USA). BioMax MS Film is from Eastman Kodak Company (NY, USA).

Derivatization of the membranes

Photolihker derivatization of the cellulose membrane is carried out as already described [41]. N ° -Fmoc-ß-Ala-OPfp is coupled to the cellulose membrane in ΝMP (FIG. 5). The remaining amino functions are then acetylated with a 10% solution of acetic anhydride in methanol. After cleavage of the Fmoc protective group, LacNAc-O- (CH ₂ ) ₈ CO ₂ H is immobilized in the presence of PfpOH, DIC and NMP. The glycine ester modification of the cellulose membrane is carried out as described in the literature [42]. LacNAc-O- (CH ₂ ) ₈ CO ₂ H is immobilized in the same way as already described for the photolinker membrane. Loading the membranes with LacNAc gave a value of 80 nmol / cm ² for the glycine ester membrane and 300 nmol / cm ² for the photolinker membrane. Appropriate membranes without LacNAc were used to determine the influence of the background.

Cleavage of the compounds from the membrane

The compounds of the glycine ester membrane were cleaved off by adding 100 μL diethylamine / water (50/50) to a cellulose strip / spot. After 1 h, 80 μL are removed and the radioactivity is determined in a scintillation counter.

The connections of the photolinker membrane were cleaved off by UV radiation of the cellulose strips / spots (each side for 3 h) at 365 nm. Then water (100 μL) is added to the strip water and after 10 min 80 μL are used for scintillation measurements.

EXAMPLE 1 α 1,3-Galactosyltransferase: Radiochemical Assay

UDP-Gal (100 mM solution in water, 5 μL, 2 mmol), UDP- [ ¹⁴ C] Gal (100,000-150,000 dpm, in 1 μL H ₂ O), α 1,3-galactosyltransferase (3.3 mU / mL, 2 μL), assay buffer (500 mM MOPS, 1 M MnCl ₂ ), AP (1 μL) and BSA (0.5 μL BSA solution, 100 mg / mL) become the immobilized acceptor (a strip of the glycine ester or photolinker membrane, size 2 x 5 mm, 8-30 nmol) in one Microcentrifuge tube added to a final volume of 230 μL. Reaction mixtures are incubated for 24 h at room temperature. The membranes are then washed successively with 1 M NaCl (15 mL, 50 ° C), buffer A (NaCl 8 g / L, KC1 0.2 g / L, Tris 6.1 g / L, pH 8.0, Tween 20 10 mL / L) (15 mL, 50 ° C), water (15 mL, 50 ° C), and EtOH (15 mL, 50 ° C). The carbohydrate derivatives are split off from the membrane and the radioactivity is quantified in a scintillation counter after adding 3.5 mL MeOH and 10 mL EcoLite (+) scintillation cocktail.

Time-dependent transfer experiment

Membrane pieces (2.0 mm diameter, 3 pieces) are mixed in with a mixture of radioactive ([ ¹⁴ C], 200,000 dpm) and non-radioactive UDP-Gal (1.2 mM) in the presence of α (l- »3) GalT (3.3 μU) incubated in a microcentrifuge tube. Six reaction vessels are prepared in this way and the reaction is stopped after 30 min, 1 h, 2 h, 3.5 h, 7.5 h and 24 h, respectively. After washing the membrane, the immobilized oligosaccharides are split off with a diethylamine / water mixture. An aliquot is taken in each case and methanol and a cocktail are added. The radioactivity of the resulting mixture is measured using a scintillation counter. The data obtained are summarized in FIG. 6.

The amount of galactose transferred increased linearly with time. The transfer rate of UDP-Gal in the presence of 3.3 μU enzyme was 12 fmol / min / mm ² . The maximum transfer was 50 pmol / mm ² , or 6.25% of the total amount of LacNAc.

Determination of the donor km

Three membrane pieces (2 mm dm) were incubated in the presence of UDP-Gal (200,000 dpm), (1-3) GalT (3.3 μU), and alkaline phosphatase (0.1 U) in microcentrifuge tubes. Eleven different concentrations of UDP-Gal were used to determine the donor Km value. After incubation, the membrane pieces were washed and the products were split off. Radioactivity was determined using a scintillation counter. The values obtained were converted into transferred gal and plotted against the UDP-gal concentration. The determined Km value is 3.1 mM (Fig. 8).

EXAMPLE 2 α 1,3-Fucosyl Transferase: Radiochemical Assay

Similarly (see Example 1), glycine esters and photo linker membranes were treated with GDP-Fuc, (1 mM solution in water, 1 µL, 25 µmol), GDP- [ ¹⁴ C] Fuc (40,000-60,000 dpm, in 2 µL H ₂ O), α 1,3-fucosyltransferase (164 mU / mL, 4 μL) and assay buffer (50 mM Tris-HCl, 20 mM MnCl ₂ , 1 mM DTT, pH 7.5, 4 μL) in a final volume of 40 μL implemented.

Time-dependent transfer experiment

Membrane pieces were incubated with radioactive and non-radioactive GDP-Fuc in the presence of α 1,3-FT (0.65 mU) for 24 h. The oligosaccharides obtained were cleaved off and the radioactivity obtained was measured by scintillation. The transfer rate of GDP-Fuc with 656 μU enzyme was determined to be 0J8 fmol / min / mm ² . The maximum transfer of GDP-Fuc was 1.1 pmol / mm ² , or 0.14% of the total amount of LacNAc (Fig. 9).

EXAMPLE 3 α 2,3- (N) -sialyltransferase: Radiochemical Assay

In a similar manner (see Example 1), glycine esters and photo linker membranes with CMP- Νeu5Ac (5 mM solution in water, 5 μL, 0.2 mM), CMP- [ ^I4 C] Neu5Ac (300,000-400,000 dpm, in 1 μL water) , α 2,3- (N) -sialyltransferase (40 mU / mL, 12.5 μL) and assay buffer (250 mM MES, 0.5% Triton CF-54, pH 7.0, 20 μL) in a total volume of 50 μL. The transfer rate of CMP-Νeu5Ac at 0.5 mU α (2-> 3) ST enzyme concentration was determined to be 6.5 fmol / min / mm ² for the glycine ester membrane and 24 fmol / min / mm for the photolinker membrane. EXAMPLE 4 α 2,6- (N) -sialyltransferase: Radiochemical Assay

In a similar way (cf. Example 1), glycine esters and photolinker membranes with CMP-Νeu5Ac (5 mM solution in water, 5 μL, 0.2 mM), CMP- [ ^{! 4} C] Neu5Ac (300,000-400,000 dpm, in 1 μL water) ), α 2,6- (N) -sialyltransferase (38 mU / mL, 5 μL) and assay buffer (250 mM Νodium cacodylate, 0.5% Triton CF-54, pH ^■ = 6.0, 20 μL) in a final volume incubated from 50 μL. The transfer rate of CMP-Νeu5Ac at an enzyme concentration of 189 μU α (2- »6) ST was 4.4 fmol / min / mm ² for the ester membrane and 65 fmol / min / mm for the photolinker membrane.

EXAMPLE 5 α 1,3-Galactosyl Transferase: Mass Spectroscopic Assay

UDP-Gal (100 mM solution in water, 5 μL, 2 mmol), α 1,3-galactosyltransferase (2 μL, 9.8 U / mL) assay buffer (500 mM MOPS, 1 M MnCl ₂ ), AP (1 μL) and BSA (0.5 μL BSA solution, 100 mg / mL) were added to the membrane-bound acceptor (a strip of the glycine ester or photolinker membrane, 2 x 5 mm, 8-30 nmol) in a microcentrifuge tube in a final volume of 230 μL. The reactions were incubated at room temperature for 24 h. The membrane pieces were washed successively with 1 M NaCl (15 mL, 50 ° C), buffer (15 mL, 50 ° C), water (15 mL, 50 ° C), and EtOH (15 mL, 50 ° C). The saccharide derivatives obtained were cleaved from the membrane, the solution was concentrated, taken up in MeOH (10 μL) and a mass spectrum was recorded using a PerSpective Biosystems ToF Mariner Biospectrometry Work Station. The spectrum obtained shows a main peak which corresponds to the unreacted acceptor LacNAc, and a further peak which corresponds to the transferase product Gal-LacNAc, formed by transfer of galactose from UDP-Gal to the membrane-bound acceptor. Similar results were obtained with the photolinker membrane.

EXAMPLE 6 α 2,3- (N) -sialyltransferase: fluorescence assay CMP-5-fluoresceinyl-neuraminic acid (0.4 mM solution in water, 20 μL, 0.2 mM), α 2,3- (N) -sialyltransferase (10 μL) and assay buffer (250 mM MES, 0.5% Triton CF54, pH 7.0 , 9.5 μL) were added to the membrane-bound acceptor (a strip of glycine ester membrane of 2 × 5 mm, 8 nmoi) in a microcentrifuge tube in a final volume of 50 μL. The reaction mixture is incubated for 4 h at room temperature and then with 1 M NaCl (15 mL, 50 ° C), buffer A (15 mL, 50 ° C), water (15 mL, 50 ° C), and EtOH (15 mL , 50 ° C). The fluorescence was measured with a transilluminator (FBTI 816, connected to a digital camera). Membrane pieces (2.0 mm in diameter) were incubated in the presence of α (2 → 3) ST (400 μU) with CMP-5-Fluoro-Neu5Ac (0.22 mM) in a microcentrifuge tube. The reaction was stopped after 2 h, 4 h and 24 h, respectively. At the same time, two negative controls (control A: without transferase, control B: LacNAc bound to glycine ester membrane) were carried out. After washing the membrane pieces, the fluorescence obtained was determined using a transilluminator (FBTI 816). The result is shown in FIG. 10. Maximum transfer was already obtained after 4 h, the two negative controls showing no fluorescence.

EXAMPLE 7 α 1,3-galactosyltransferase: autoradiography assay

UDP-Gal (100 mM solution in water, 5 μL, 2 mmol), UDP- [ ^{! 4} C] -GaI (100,000-150,000 dpm, in 1 μL H ₂ O), 1,3-galactosyltransferase (2 μL) assay buffer (500 mM MOPS, 1 M MnCl ₂ ), AP (1 μL) and BSA (0.5 μL BSA solution, 100 mg / mL) became the membrane-bound acceptor (a strip of the glycine ester or photolinker membrane, 2 5 mm, 8-30 nmol) placed in a microcentrifuge tube in a final volume of 230 μL. The reactions were incubated at room temperature for 24 h. The membrane pieces were washed successively with 1 M NaCl (15 mL, 50 ° C), buffer A (15 mL, 50 ° C), water (15 mL, 50 ° C), and EtOH (15 mL, 50 ° C). For exposure, the membrane pieces are placed on a Kodak BioMaxMS film for 2 days at -70 ° C. Membrane pieces (2 x 5 mm) were incubated with radioactive UDP-Gal ([C ¹⁴ ], 200,000 dpm) in a microcentrifuge tube for 24 h. Three different concentrations of α (1-3) GalT, 132 μU, 13.2 μU and 1.32 μU were incubated with both the glycine ester and the photolinker membranes. The radioactivity of all membrane pieces was determined by autoradiography (Kodak BioMaxMS film, -70 ° C, 6 days exposure time). The results obtained are shown in Fig. 11.

It turned out that the negative controls (membranes without LacNAc) showed no radioactivity. The amount of radioactivity transferred is proportional to the concentration of the enzyme used. The photolinker membranes generally showed higher transfer rates.

EXAMPLE 8 α 1,3-Fucosyl Transferase: Autoradiography Assay

In a similar way (see Example 7), glycine esters and photolinker membranes with GDP-Fuc (ImM solution in water, 1 μL, 25 μmol), GDP- [ ¹⁴ C] -Fuc (40,000-60,000 dpm, in 2 μL H ₂ O), α 1,3-fucosyltransferase (164 mM, 4 μL) and assay buffer (50 mM Tris-HCl, 20 mM MnC12, 1 mM DTT, pH 7.5, 4 μL) in a final volume of 40 μL.

EXAMPLE 9 α 2,3- (N) -sialyltransferase autoradiography assay

In a similar manner (cf. Example 7), glycine esters and photolinker membranes with CMP-Νeu5Ac (5 mM solution in water, 5 μL, 0.2 mM), CMP- ¹⁴ C-Neu5Ac (300,000-400,000 dpm, in 1 μL water), α 2,3- (N) -sialyltransferase (40 mM, 12.5 μL) and assay buffer (250 mM MES, 0.5% Triton CF54, pH 7.0, 20 μL) in a final volume of 50 μL.

EXAMPLE 10 α 2,6- (N) -sialyltransferase autoradiography assay In a similar manner (cf. Example 7), glycine esters and photolinker membranes with CMP-Neu5Ac (5 mM solution in water, 5 μL, 0.2 mM), CMP- ¹⁴ C-Neu5Ac (300,000-400,000 dpm, in 1 μL water), α 2,6- (N) -sialyltransferase (38 mM, 5 μL) and assay buffer (250 mM sodium cacodylate, 0.5% Triton CF-54, pH = 6, 20 μL) in a final volume of 50 μL.

The features of the invention disclosed in the above description, the claims and the drawings can be essential both individually and in any combination for realizing the invention in its various embodiments.

Claims

Expectations

1. A method for detecting an enzymatic glycosyltransferase activity in a sample, wherein

the sample is reacted together with a glycosyl donor and a glycosyl acceptor immobilized on a carrier, and

a transferase product is detected in the presence of an enzymatic glycosyltransferase activity,

characterized in that the carrier is a cellulose membrane.

2. The method according to claim 1, characterized in that the glycosyl donor is present in excess concentration, in particular the excess corresponds at least to twice the K value of the glycosyltransferase to be detected.

3. The method according to claim 1 or 2, characterized in that the glycosyl acceptor is selected from the group comprising monosaccharides, oligosaccharides, in particular part of an oligosaccharide, polysaccharides and glycoconjugates such as glycoproteins, peptides and lipids.

4. The method according to claim 3, characterized in that the glycoconjugate is a lactosamine derivative, in particular the glycoconjugate LacNAc.

5. The method according to any one of claims 1 to 4, characterized in that the glycosyl acceptor is immobilized on the membrane directly or via a linker system.

6. The method according to claim 5, characterized in that the linker system is selected from the group consisting of light-cleavable linkers (photolinker) and carboxylic ester linkers, in particular glycine ester linkers.

7. The method according to any one of claims 1 to 6, characterized in that the glycosyl donor is a nucleotide sugar.

8. The method according to claim 1, characterized in that the nucleotide sugar is selected from the group consisting of UDP-Gal, UDP-GalNAc, UDP-Glc, UDP-GlcNAc, UDP-Xyl, UDP-GluA,, GDP-Man, GDP- Fuc and CMP-NeuAc.

9. The method according to any one of claims 1 to 8, characterized in that the glycosyl donor carries a label or a chemoselective function that enables later labeling.

10. The method according to claim 9, characterized in that the label is selected from the group comprising fluorescent labels, radioactivity labels, enzyme activity labels and chemiluminescence labels.

11. The method according to any one of claims 1 to 10, characterized in that the glycosyltransferase is selected from the group consisting of galactosyltransferases, fucosyltransferases, sialyltransferases, glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases and glucuronosyltransferases and glucuronosyltransferases.

12. The method according to claim 11, characterized in that the galactosyltransferase is a beta-1,3- or beta-1,4-galactosyltransferase.

13. The method according to claim 11, characterized in that the fucosyltransferase is selected from the group comprising alpha-l, 3/4 fucosyltransferases and alpha-l, 2-fucosyltransferases -V.

14. The method according to claim 11, characterized in that the sialyltransferase is selected from the group comprising alpha-2,3-sialyltransferases and alpha-2,6-sialyltransferases.

15. The method according to any one of claims 1 to 14, characterized in that the sample is selected from the group comprising human, animal, vegetable and microbial samples.

16. The method according to claim 15, characterized in that the sample is selected from the group comprising tissue, in particular tissue containing Golgi apparatus, and body fluids.

17. The method according to claim 15, characterized in that the sample is selected from the group comprising cell lysates, tissue lysates, organ lysates, extracts, crude extracts, purified preparations and unpurified preparations.

18. The method according to claim 16, characterized in that the body fluid is selected from the group comprising breast milk, urine, sputum, saliva, serum and lymphatic fluid.

19. A method for the parallel detection of the enzymatic activity of glycosyltransferase (s) in a large number of samples, characterized in that the method according to one of claims 1 to 18 is carried out in parallel for each sample.

20. A method for determining the glycosyltransferase activity pattern of a sample, characterized in that

reacting an aliquot of a sample together with a first combination of a glycosyl donor and a glycosyl acceptor immobilized on a carrier and determining whether a transferase product is formed, and

another aliquot of a sample is reacted together with a second combination of a glycosyl donor and a glycosyl acceptor immobilized on a carrier and it is determined whether a transferase product is formed, and

optionally the transferase product is determined, characterized in that the carrier is a cellulose membrane.

21. A method for determining the substrate specificity of a glycosyltransferase, characterized in that

the glycosyltransferase is provided in a sample,

reacting an aliquot of the sample together with a first combination of a glycosyl donor and a glycosyl acceptor immobilized on a carrier and determining whether a transferase product is formed, and

another aliquot of the sample is reacted together with a second combination of a glycosyl donor and a glycosyl acceptor immobilized on a carrier and it is determined whether a transferase product is formed, and

optionally the transferase product is determined,

characterized in that the carrier is a cellulose membrane.

22. Use of a method according to one of the preceding claims for the diagnosis and / or monitoring of diseases which are associated with an altered enzymatic glycosyltransferase activity.

23. Use according to claim 22, characterized in that the disease is selected from the group comprising cirrhosis of the liver, immune deficiency diseases, lysosomal storage diseases and tumor diseases.

24. Use according to claim 23, characterized in that the tumor disease is selected from the group comprising human carcinomas, in particular melanomas, breast, colon, chorion, liver cell and bladder carcinomas, and animal carcinomas.

25. The method according to any one of the preceding claims, characterized in that the individual reaction steps are carried out in parallel, preferably highly parallel.

26. A method for identifying a compound influencing the expression of a glycosyltransferase comprising the following steps:

Providing a candidate connection,

Testing the candidate compound for its ability to affect the expression of a glycosyltransferase in an expression system, wherein

the expression of the glycosyltransferase is determined in that a glycosyl acceptor immobilized on a support and a glycosyl donor are present in the expression system,

and the presence or absence of a transferase product is demonstrated,

characterized in that the carrier is a cellulose membrane.

27. The method according to claim 26, characterized in that the compound inhibits the expression of the glycosyltransferase.

28. The method according to claim 26 or 27, characterized in that the expression is the transcription.

29. The method according to claim 26 or 27, characterized in that the expression is translation.

30. A method for identifying a compound influencing the activity of a glycosyltransferase comprising the following steps:

Providing a candidate connection, Testing the candidate compound for its ability to affect the activity of a glycosyltransferase in a reaction batch, wherein

the activity of the glycosyltransferase is determined by the presence of a glycosyl acceptor immobilized on a support and a glycosyl donor in the reaction mixture,

and the presence or absence of a transferase product is demonstrated,

characterized in that the carrier is a cellulose membrane.

31. The method according to claim 30, characterized in that the compound inhibits the activity of the glycosyltransferase.

32. The method according to any one of claims 26 to 31, characterized in that the substeps of the method are carried out in parallel with at least two different candidate compounds.

33. The method according to any one of claims 26 to 32, characterized in that the candidate compound is contained in a library of compounds, in particular a library of low molecular weight compounds.

34. Kit for the detection of an enzymatic glycosyltransferase activity comprising at least one glycosyl acceptor immobilized on a support, characterized in that the support is a cellulose membrane.

35. Kit according to claim 34, characterized in that the kit comprises a glycosyl donor.

36. Kit according to claim 34 or 35 comprising at least one further component which is selected from the group comprising buffers, glycosyl donor (s), glycosyl acceptor (s) and glycosyltransferase ^).