DE10122452A1 - Method for the detection of the enzymatic activity of glycosyltransferases - Google Patents

Abstract

The present invention relates to a method for detecting an enzymatic glycosyltransferase activity in a sample, wherein DOLLAR A - the sample is reacted together with a glycosyl donor and a glycosyl acceptor immobilized on a support, in particular a cellulose membrane, and DOLLAR A - in the presence of an enzymatic glycosyltransferase Activity of a transferase product is detected.

Description

The invention relates to a method for the detection of the enzymatic glycosyltransferase Activity in a sample, a method for determining glycosyltransferase activity Pattern of a sample, a method for determining the substrate specificity of a Glycosyltransferase, use of the aforementioned methods in the context of diagnosis of diseases, a method of identifying the expression of a Glycosyltransferase influencing compound and a method for identifying a enzymatic activity of a compound influencing glycosyltransferase and a kit for the Detection of the enzymatic activity of a glycosyltransferase.

Glycosyltransferases transfer activated mono- or oligosaccharides from glycosyl donors on glycosyl acceptors. Mono-, oligo- or polysaccharides, peptides, Proteins or lipids, as well as their combinations, are then used as glycoconjugates available. Glycoconjugates play a key role in many biological processes. So they act as receptor sites for enzymes, hormones, antibodies, lectins, toxins, as well for microorganisms such as viruses and bacteria. Physiologically and pathophysiologically The involvement of glycoconjugates in the control of cell functions is particularly important such as cell growth, cell-cell recognition, expression of antigens, cell adhesion, infection, as well as cell differentiation. The growth-dependent pattern of glycosylation of the Glycoproteins are mainly determined by the activity and specificity of those in the Golgi apparatus localized glycosyltransferases.

Changes in the activity and expression of specific glycosyltransferases are available with the Tumor development and other disease conditions closely related. To the An example is the changed composition of the cell surface oligosaccharides in direct Relationship with degree of transformation and metastasis formation (Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M.L., Kerbel, R.S., Science 1987, 236, 582). Malignant Changes in mouse and human cells generally go with expression more complex N-glycosidic structures as well as higher polylactosamine content (Warren, L., Buck, C.A., Tuszynski, G.P., Biochem. Biophys. Acta. 1978, 516, 97). Latest Studies bring the branching and the polylactosamine content of the O-oligosaccharides with Tumor development and metastasis formation in connection (Yousefi, S., Higgins, E., Daoling, Z., Pollex-Kruger, A., Hindsgaul, O. and Dennis, J.W., 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 Binding (α or β), and the hydroxyl group of the acceptor involved on the Monosaccharide is transferred. With few exceptions, transferases catalyze highly regional and stereo-selective, which is why around 200 different glycosyltransferases must exist in order to to enable all known oligosaccharide structures (Paulson, J.C., and Colley, K.J., J. Biol. Chem. 1989, 264, 17615). The recognition of the acceptor substrate is only based on this on a few hydroxyl groups, the so-called "polar key groups". This could already on numerous protein-carbohydrate interactions like with lectins, Antibodies, glycosidases and oxidases are shown (Lemieux, R. U., Acc. Chem. Res. 1996, 29, 373).

The α1,3-galactosyltransferase (α1,3-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 Galβ1 → 4GlcNAc-R ( Fig. 1 ) and thus responsible for the formation of Galα1 → 3Galβ1 → 4GlcNAc epitopes, which occur on glycoproteins and glycolipids, has recently become increasingly important (Joziasse, DH, Shaper, NL, Salyer, LS, Van den Eijnden, DH , by Spoel, AC, Shaper, JH, Eur. J. Biochem. 1990, 191, 75; Fang, J., Li, J., Chen, X., Zhang, Y., Wang, 3., 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 Galα1 → 3Galβ1 → 4GlcNAc epitope is the most important antigen which is responsible for the acute rejection of transplants (Galili, U., Immunol. Today 1993, 14, 480; Joziasse, DH, Oriol, R., Biochim. Biophys Acta 1999, 1455, 403). Recombinant α1,3-GalT and a fusion protein consisting of UDP-Gal epimerase and α1,3-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 α1,3-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 Galβ1 → 3GlcNAc, Galβ1 → 4Glc (Sujino, K., Malet, C., Hindsgaul, O., Palcic, MM, Carbohydr. Res. 1998, 305, 483; Blanken, WM, von 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 the 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, 5th J., Stults, C.L. , Srnka, CA, Glycobiology 1991, 1, 577; Hakomori, S.-I, 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 β-D-galactose-containing structures to form the Le ^a and Le ^x blood group antigens (Macher , BA, Holmes, EH, Swiedler, SJ, Stults, C. L, Srnka, CA, Glycobiology 1991, 1, 577; Natsuka, S., Lowe, 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 α1,3 / 4-fucosyltransferase and α1,3-fucosyltransferase are found in breast milk. Five different human α1,3 / 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 α1,3 / 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 Galβ1 → 3GlcNAc (type I) and Galβ1 → 4GlcNAc ( Type II) structures are catalyzed in order 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 α1,3 / 4-FucT from breast milk, since both type I and type II acceptors are used as substrates (cf. FIG. 2). FucT V is an important industrial enzyme that has already been used in the industrial production of Sialyl Lewis ^x .

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, JC, 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, JC, 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 (Kelm, 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, JC, 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 III) from rat liver transfers neuraminic acid from CMP-neuraminic acid to the 3-OH function of terminal Gal residues in Galβ1 → 4GlcNAc or Galβ1 → 3GlcNAc 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 Galβ1 → 4GIcNAc (cf. FIG. 4 ) (Harduin-Lepers, A., Recchi, M.-A., Delannoy, P., Glycobiology 1995, 5, 741; Tsuji, S., Datta, AK, Paulson, JC, 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 using 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 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, TA, Oppenheimer, CL, Paulson, JC, Prieels, J.-P., Rearick, J. L, 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, N. 5., Arch. Biochem. Biophys. 1990, 283, 372; White, AR, Xin, Y. , Pezeshk, V., Bioche M. 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. Here, 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, SC, Hindsgaul, O., Alton, G., Pierce, M., Palcic, MM , Analt. Biochem. 1990, 185, 112; Gosselin, S., Alhussaini, M., Streift, MB, Takabayashi, K., Palcic, MM, Analt. Biochem. 1994, 220, 92; Shedletzky, E., Unger , C., Delmer, DP, Anall. Biochem. 1997, 249, 88; Kodama, H., Kajihara, Y., Endo, T., Hashimoto, H., Tetrahedron Lett. 1993, 34, 6419).

Disadvantage of the test methods previously available for the investigation of the enzymatic Glycosyltransferase activity is their limited sensitivity and specificity as well as their inadequate usability for screening a large number of samples (high throughput system = HTS) as it is for a diagnostic application, for example for the diagnosis of Tumors (prevention, 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 To provide glycosyltransferase activity which has the disadvantages mentioned above State-of-the-art method overcomes and in particular also in the HTS and in the Diagnosis of diseases can be used.

• - die Probe zusammen mit einem Glycosyldonor und einem Träger, insbesondere einer Zellulosemembran, immobilisierten Glycosylakzeptor umgesetzt wird, und
• - bei Anwesenheit einer enzymatischen Glycosyltransferase-Aktivität ein Transferaseprodukt nachgewiesen wird.

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 with a glycosyl donor and a carrier, in particular a cellulose membrane, immobilized glycosyl acceptor, and
• - A transferase product is detected in the presence of an enzymatic glycosyltransferase activity.

In one embodiment it is provided that the glycosyl donor is present in excess is. In a preferred embodiment it is provided that the excess is at least corresponds to 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 a 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-acetyl lactosamine derivative.

In one embodiment, the glycosyl acceptor is provided directly or via a Linker system is immobilized on the membrane.

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 are very particularly preferred Glycinesterlinker.

In a further embodiment it is provided that the glycosyl donor is a Is nucleotide sugar.

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

In one embodiment it is provided that the glycosyl donor is a label or a carries chemoselective function, this preferably a later marking allows.

In a preferred embodiment it is provided that the marking is selected from the group that uses fluorescent labels, radioactivity labels, enzyme activity Includes labeling and chemiluminescent labels.

In a further embodiment it is provided that the glycosyltransferase is selected from the group consisting of galactosyltransferases, fucosyltransferases, sialyltransferases, Glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, Glucuronyltransferases and glucosamine transferases.

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

In an alternative preferred embodiment it is provided that the Fucosyltransferase is selected from the group consisting of alpha-1,3 / 4-fucosyltransferases and alpha-1,2- Fucosyltransferases includes.

In a further alternative preferred embodiment it is provided that the Sialyltransferase is selected from the group consisting of alpha-2,3-sialyltransferases and alpha-2,6- Includes sialyltransferases.

In one embodiment it is provided that the sample is selected from the group that includes human, animal, vegetable and microbial samples.

In a preferred embodiment it is provided that the sample is selected from the Group that includes tissues and body fluids.

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

In a further embodiment it is provided that the sample is selected from the Group, 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 consisting of breast milk, urine, sputum, saliva, serum and Lymphatic fluid includes.

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 variety of samples, characterized in that for each sample the invention Procedure is carried out in parallel.

• - ein Aliquot einer Probe zusammen mit einer ersten Kombination aus einem Glycosyldonor und einem an einem Träger, insbesondere einer Zellulosemembran, immobilisierten Glycosylakzeptor umgesetzt wird und bestimmt wird, ob ein Transferaseprodukt gebildet wird, und
• - ein weiteres Aliquot einer Probe zusammen mit einer zweiten Kombination aus einem Glycosyldonor und einem an einem Träger, insbesondere einer Zellulosemembran, immobilisierten Glycosylakzeptor umgesetzt wird und bestimmt wird, ob ein Transferaseprodukt gebildet wird.

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
• a further 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 to determine the transferase product, the determination of the transferase product preferably its biological and / or chemical and / or physical Characterization includes.

• - die Glycosyltransferase in einer Probe bereitgestellt wird,
• - ein Aliquot der Probe zusammen mit einer ersten Kombination aus einem Glycosyldonor und einem an einem Träger, insbesondere einer Zellulosemembran, immobilisierten Glycosylakzeptor umgesetzt wird und bestimmt wird, ob ein Transferaseprodukt gebildet wird, und
• - ein weiteres Aliquot der Probe zusammen mit einer zweiten Kombination aus einem Glycosyldonor und einem an einem Träger, insbesondere einer Zellulosemembran, immobilisierten Glycosylakzeptor umgesetzt wird und bestimmt wird, ob ein Transferaseprodukt gebildet wird.

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
• a further 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 to determine the transferase product, the determination of the transferase product preferably its biological and / or chemical and / or physical Characterization includes.

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 associated with an altered glycosyltransferase activity.

In one embodiment it is provided that the disease is selected from the group liver cirrhosis, immune deficiency diseases, lysosomal storage diseases and Tumor diseases include.

In a preferred embodiment it is provided that the tumor disease is selected is from the group that includes human and animal cancers.

In a particularly preferred embodiment it is provided that the carcinoma is selected from the group consisting of melanomas, breast cancers, colon cancers, Choriocarcinoma, hepatoma carcinoma and bladder cancer.

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

An application of this embodiment is a high throughput system in which for a variety of reaction approaches simultaneously detecting the enzymatic Glycosyltransferase activity, the determination of the glycosyltransferase pattern and / or the The substrate specificity of a glycosyltransferase is determined.

• - Bereitstellen einer Kandidaten-Verbindung,
• - Testen der Kandidaten-Verbindung hinsichtlich ihrer Fähigkeit, die Expression einer Glycosyltransferase in einem Expressionssystem zu beeinflussen, wobei
• - die Expression der Glycosyltransferase bestimmt wird, indem ein auf einem Träger, insbesondere einer Zellulosemembran, immobilisierter Glycosylakzeptor und ein Glycosyldonor in dem Expressionssystem vorhanden sind,
• - und die Anwesenheit oder Abwesenheit eines Transferase-Produktes nachgewiesen wird.

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 glycosyl transferase 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 demonstrated.

In one embodiment it is provided that the compound expresses the Glycosyltransferase inhibited.

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.

• - Bereitstellen einer Kandidaten-Verbindung,
• - Testen der Kandidaten-Verbindung hinsichtlich ihrer Fähigkeit, die Aktivität einer Glycosyltransferase in einem Reaktionsansatz zu beeinflussen, wobei
• - die Glycosyltransferase-Aktivität bestimmt wird, indem ein auf einem Träger, insbesondere einer Zellulosemembran, immobilisierter Glycosylakzeptor und ein Glycosyldonor in dem Reaktionsansatz vorhanden sind,
• - und die Anwesenheit oder Abwesenheit eines Transferase-Produktes nachgewiesen wird.

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 influence the activity of a glycosyltransferase in a reaction mixture, wherein
• 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 demonstrated.

In one embodiment it is provided that the compound has the activity of Glycosyltransferase inhibited.

In a further embodiment it is provided that the sub-steps of the method for Identification of a compound influencing the expression of a glycosyltransferase and / or the substeps of the method for identifying the activity of a Glycosyltransferase influencing compound in parallel with at least two different Candidate connections are carried out.

In yet another embodiment of the method according to the invention, 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 immobilized on a support, preferably a cellulose membrane Glycosyl.

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

In a further embodiment it is provided that the kit has at least one further one Component which is selected from the group consisting of buffers, glycosyl donor (s), Glycosyl acceptor (s) and glycosyltransferase (s) includes.

The present invention is based on the surprising finding that with the Immobilization of a glycosyl acceptor on a support, in particular a membrane and preferably a cellulose membrane, a highly sensitive process for Investigation of the activity of glycosyltransferases in the broadest sense is available. The high sensitivity and selectivity of the method allows the determination of the Glycosyltransferase activities for all those cases where the sample material is limited is available, such as in raw extracts. At the same time, there is an advantage with inventive method in that precisely because of this high sensitivity Procedure for high parallel analysis procedures (English high throughput screening) is suitable. The Investigation of the enzymatic activity of glycosyltransferases can be done in different aspects can be divided. One aspect of the method concerns the detection of an or the enzymatic glycosyltransferase activities in a sample, a second aspect the Determination of the glycosyltransferase activity pattern of a sample and another aspect Determination of the substrate specificity of a glycosyltransferase. All of these procedures are The aforementioned use of the immobilized glycosyl acceptor is common and causes the surprising advantages of the system.

The term detection of glycosyltransferase activity in a sample describes herein generally evidence and includes both a qualitative and a quantitative detection of glycosyltransferase activity. The type of glycosyltransferase involved in The approach chosen can depend on the one used Glycosyl donor or the immobilized glycosyl acceptor and the other of the selected Reaction conditions that are selected in the detection method. It is in the frame the knowledge of the person skilled in the art, the possible glycosyl donors which possible glycosyl acceptors and the specific reaction conditions for each Glycosyltransferase activity to be detected and thus the specificity of the To ensure detection of a specific glycosyltransferase activity.

When determining the glycosyltransferase activity pattern of a sample, it will assumed that one or more glycosyltransferases are contained in the sample. By Can use different combinations of glycosyl donor and glycosyl acceptor the enzymatic activities of various glycosyltransferases are determined. It is also within the scope of the present invention, various combinations of Glycosyl donor (s) and glycosyl acceptor (s) to be used with the aim of being without knowledge of the specific combination (s) for the individual glycosyltransferase to be detected Activity (s), a combination pattern consisting of the parameters Generate glycosyl donor and glycosyl acceptor. Such a pattern can be used for a certain sample can be quite specific and the recognition of a certain one Condition, e.g. B. a disease state, or to identify the type of a specific sample allow without the actual specificities of those contained in the sample Glycosyltransferase must necessarily be known.

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

In the inventive method described herein, the glycosyltransferase is typically contained in a sample or the sample is one in which a Glycosyltransferase is present. Typically, a predetermined amount of sample is added labeled glycosyl donor and that used on the preferred Cellulose membrane immobilized glycosyl acceptor contacted. The right ones Reaction conditions are determined with regard to the glycosyltransferase to be examined and the one present Glycosyl donors selected. 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 are effective Reaction of one or the glycosyltransferase with the labeled glycosyl donor and the Allow glycosyl acceptor. The selection of the appropriate reaction conditions is within the framework the knowledge of the specialist in this field and is particularly in the event that a specific glycosyltransferase to be determined in a sample, known from this or easy to determine. The usual incubation times are 5 minutes to 24 Hours. The pH value and the temperature can, not least depending on the Glycosyltransferase to be detected, can be located in a wide range. Typical pH values are pH 5 to 8, the typical temperatures are 20 ° C to 45 ° C. The amount of used glycosyl donor and immobilized glycosyl acceptor depends on the investigating glycosyltransferase. Preferably, the concentration of the Glycosyl donors at least twice the km of the glycosyl transferase to be investigated amount, 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 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 reaction by simply washing the cellulose membrane with buffer solution, such as for example PBS. The content of the Glycosyltransferase in the sample determined from the amount of transferase product obtained become.

The transferase product is typically the result of Glycosyltransferase activity modified glycosyl acceptor, which is derived from the glycosyl donor Group or part of the molecule. Basically, however, it is also within the Present invention that instead of the transferase product also due to the Glycosyltransferase measured change in the glycosyl donor is measured, d. H. the Glycosyltransferase 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 resulting by the residue or part of the molecule transferred by the glycosyltransferase 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.

In the broadest sense, a "transferase product" can also include a glycosyl donor can be understood, a group or a due to the glycosyltransferase activity Part of the molecule was transferred to a glycosyl acceptor. Furthermore, here under the The term glycosyltransferase understood any enzymatic activity that leads to a Glycosyl transfer leads. The term "glycosyltransferase" is used synonymously herein 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 has further enzymatic activities.

The method chosen for determining the labeled transferase product depends on the type of labeling of the glycosyl donor or 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. Autoradiography could, for example, be indicated in particular if the method for the detection of an enzymatic or the 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 at distinct locations on the biochip differ with the consequence that different transferase products can arise.

As provided in one embodiment, cleavable linker systems can be between Glycosyl acceptor and the carrier, in particular the cellulose membrane, can be used. As a result the cleavability, the transferase products can be cleaved from the membrane and then by conventional analysis techniques such as mass spectroscopy, NMR, DC, HPLC etc. can be demonstrated. The used in the process of the invention Detection methods are known to those skilled in the art, as set forth above. In addition to the detection methods mentioned above, it is also possible to use Mass change of the immobilized glycosyl acceptor a transferase activity or a Transferase product in the form of the immobilized modified as a result of the glycosyltransferase activity Detect the glycosyl acceptor by means of plasmon resonance, since in plasmon resonance minimal mass change of approx. 180 Da is required and therefore this technique for Detection of a transfer of monosaccharides by the glycosyltransferase to be detected is applicable.

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, 506-5 13). 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 imidazoles 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 oximes from amino-oxy compounds and ketones, from hydrazone from hydrazines and aldehydes, from Hydrazone from hydrazide (see Fig. 7).

Immobilization of the substrates used, i. H. of glycosyl acceptors happens by forming a covalent bond of the saccharide or glycoconjugate with the Surface of the carrier, especially the cellulose surface. The substrates can directly, e.g. B. by reaction of an aldehyde function of the immobilizing, reducing Saccharides with an α-functionalized cellulose surface, through reductive Amination or via suitable spacers (English: spacer) linker, which is a later cleavage enable to be bound to the carrier, in particular to the cellulose membrane. As Linkers are particularly suitable for systems that operate under both mild conditions attachable as well as cleavable, d. 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 linker (R. Frank, Tetrahedron 1992, 48, 9217). An overview of common and applicable to the present invention and usable linker systems are given, for example, in B.A. Bunin, The Combinatorial Index, Academic Press, 1998, as well as in D. Obrecht, J.M. Villalgordo, Combinatorial and parallel synthesis of small-molecular-weight compound libraries, Elsevier, 1998.

Due to the fact that the immobilized glycosyl acceptor can be split off, the Method 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, a step in which the transferase product or a part thereof from that Carrier or the cellulose membrane is split off.

Within the scope of the methods according to the invention described herein, a wide variety can be used immobilized glycosyl acceptors can be used. Both monosaccharides and oligosaccharides and polysaccharides also 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 glycosyl transferases within the scope 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, GW Hart, P. Sina ≙, Carbohydrates in Chemistry and Biology 2000, Wiley-VCH, 689-624)

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

In the method according to the invention for determining the glycosyltransferase activity Pattern can be provided that the aliquot of a sample, together with a first Combination of a glycosyl donor and one immobilized on a support Glycosylak acceptor is implemented, continues to be used and so far another aliquot of one Represents sample. It is also within the scope of the present invention that the sample between the various steps are further processed, for example by specific Removal or inhibition of compounds contained in the sample or in the reaction mixture or enzymatic activities, such as that of using the first combination examined glycosyltransferase. 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 involve 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; 5. Hakamori, Y. Zhang, Chem. Biol. 1997, 4, 97; 5. J. Danishefsky, JR Allen, Angew. Chem. Int. Ed. 2000, 39, 836-863) on the Cell surface of the tumor cells can be detected, which do not occur in normal tissue or only 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 ^a , Le ^b 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 different structures based on the different disease states underlying expression or activity patterns of glycosyltransferases decrease, these be assigned.

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. Table 2

References to Table 2 1 WW Young, HS Johnson, Y. Tamura, K.-A. Karlsson, G. Larson, JMR Parker, DP Khare, U. Spohr, DA Baker, O. Hindsgaul, RU Lemieux, J. Biol. Chem. 1983, 258, 4890-4894.
2 M. Blaszczyk, GC Hansson, K.-A. Karlsson, G. Larson, N. Strömberg, J. Thurin, M. Herlyn, Z. Steplewski, H. Koprowski, Arch. Biochem. Biophys., 1984, 233, 161-168.
3 JL 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. Führer, Somat. Cell Genet. 1979, 5, 957-972.
5 D. Chia, PI Terasaki, N. Suyama, J. Galton, M. Hirota, D. Katz, Cancer Res. 1985, 45, 435-437.
6 MR Stroud, SB Levery, MEK Salyan, CE Roberts, S. Hakamori, Eur. J. Biochem. 1992, 203, 577-586.
7 H. Ito, K. Tashiro, MR Stroud, TF ∅rntoff, P. Meldgaard, AK Singhal, S. Hakamori, Cancer Res. 1992, 52, 3739-3745.
8 K. Abe, JM McKibbin, S. Hakamori, J. Biol. Chem. 1983, 258, 11793-11797.
9 A. Brown, T. Feizi, HC Gooi, MJ Embleton, JK Picard, RW Baldwin, Biosci. Reports 1983, 3, 163-170.
10 KO Lloyd, G. Larson, N. Strömberg, J. Thurin, K. -A. Karlsson, Immunogenetics 1983, 17, 537-541.
11 T. Kaizu, SB Levery, ED Nudelman, RE Stenkamp, 5th Hakamori, J. Biol. Chem. 1986, 261, 11254-11258.
12 ED Nudelman, SB Levery, T. Kaizu, S. Hakamori, J. Biol. Chem. 1986, 261, 11247-11253.
13 K. Fukushima, M. Hirota, PI Terasaki, A. Wakisaka, H. Togashi, D. Chia, N. Suyama, Y. Fukushi, ED Nudelman, S. Hakamori, Cancer Res. 1984, 44, 5279-5285.
14 Y. Fukushi, ED Nudelman, SB 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:
Table 3

References to Table 3 15a) M. Pierce, J. Arango, J Biol. Chem. 1986, 261, 10772-10777.
b) JW Dennis, S. Laferte, C. Waghorne, ML Breitman, RS Kerbel, Science 1987, 236, 582-585.
c) K. Yamashita, T. Ohkura, Y. Tachibana, S. Takasaki, A. Kobata, 1 Biol. Chenz 1984, 259, 10834-10840.
d) JW Dennis, K. Kosh, D. -M. Bryce, ML 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.
17a) S. Narashima, H. Schachter, S. Rajalakshmi, J. Cell Biol. 1988, 263, 1273-1281.
b) 18e)
18a) SH Itzkowitz, M. Yan, Y. Fukushi, A. Palekar, PC Phelphs, AM Shamsuddin, BF Trump, S. hakamori, YS Kim, Cancer Res. 1986, 46, 2627-2632.
b) S.-I. Hakomori, Adv. Cancer Res. 1989, 52, 257-331.
c) KD Lloyd, Am. J. Clin. Pathol. 1987, 87, 129-139.
19a) S. Yousefi, E. Higgins, Z. Doaling, O. Hindsgaul, A. Pollex-Kruger, JW Dennis, J. Biol. Chem. 1991, 266, 1772-1783.
b) K. Shimodaira, J. Nakayama, N. Nakamura, O. Hasebe, T. Katsuyama, M. Fukuda, Cancer. Res. 1997, 57, 5201-5206.

As a result of this and the principle using the method according to the invention determinable substrate specificities or glycosyltransferase activity patterns can be a effective diagnostic, follow-up and prognostic system. Under Follow-up control is understood here in particular that before the start of therapy the corresponding glycosyltransferase activity, the glycosyltransferase pattern or the Substrate specificity of the glycosyltransferase is determined and repeated in the course of a treatment Samples are taken from the treated individual and based on these parameters be examined. Is there a correlation between the corresponding parameters and the course of the disease, for example by determining the activity of certain Glycosyltransferases with worsening or worsening of the disease Tumor growth goes hand in hand with the determined parameters as a function of Appropriate conclusions can be drawn during the course of treatment.

The same applies in principle to the prognostic application of such systems, whereby here under forecast especially statements regarding the probability of recovery of a patient, whether under the influence of treatment or without. In addition to the correlation described above, there is also a more extensive one Correlation with the respective parameters such that a statement from the course is required the parameters or their change or non-change over time with the Probability of recovery, stabilization of a condition or continuation of one Condition or deterioration of the condition of one or the individual exists.

In a further aspect, the invention relates to methods for identifying a Expression of a compound influencing glycosyltransferase. The concept of expression is to be understood here in particular in such a way that it concerns both the transcription translation can act as well. As a result of using an immobilized Glycosyl acceptor in combination with a glycosyl donor and a glycosyltransferase can this method is also particularly effective in terms of use in a high throuput System (HTS) can be designed. In the compounds influencing expression it is preferably inhibitors. The inhibitors can be transcription or reduce or completely prevent the translation of the glycosyltransferase. With the through the specific procedure, in particular the use of the immobilized Glycosyl acceptor on a cellulose membrane, increased sensitivity of the procedure can therefore also make minor changes to the expressed glycosyltransferase are detected and a differentiation of the different inhibitors is made become. The same applies in principle to those compounds which express the expression of a Increase glycosyltransferase. The compound influencing expression is also used herein referred to as a candidate connection, especially if it is for the first time in a Such test system is used and it is not known if and if so, in which Extent that affects the expression of a glycosyltransferase. As related to The other methods of the present invention disclosed herein can use glycosyltransferase practically any glycosyltransferase, but is preferably such as they in connection with the other methods of the invention described herein is used. The same applies to the glycosyl donors and glycosyl acceptors and the respective process parameters.

In the method according to the invention for identifying an expression influencing connection, said connection can be either transcription or translation influence, d. H. inhibit or stimulate, said compound is preferably an inhibitor.

After in the above method, the features of the invention also Procedures for the detection of a glycosyltransferase are included and for assessment the question of whether, and if so, to what extent a candidate connection actually expresses ultimately inhibits or stimulates the glycosyltransferase activity in the reaction mixture Further steps are typically required to determine on what level of expression the candidate compound is actually in the reaction process intervenes. This can be done, for example, by examining whether, and if so, to what extent the mRNA coding for the respective glycosyltransferase in the Expression system is present. Is the amount of mRNA under the influence of the candidate Compound decreased with decreased glycosyltransferase activity, of which be assumed that the inhibitory effect of the candidate compound at the level of Transcription takes place. The methods for determining the amount of mRNA are the Known to those skilled in the art. The same considerations apply analogously to the case that the candidate compound has a stimulating effect.

Conversely, one can decrease under the influence of a candidate connection Glycosyltransferase activity and at the same time unchanged amount of mRNA thereof be assumed that the inhibitory effect of the candidate compound at the level the translation takes place. Under such conditions, the amount would be too translated, the glycosyltransferase-containing protein usually reduced. The proceedings to determine the amount of translated protein containing the glycosyltransferase are known to those skilled in the art. Examples are antibodies against that said protein referred. The same considerations apply mutatis mutandis in the event that the Candidate compound has a stimulating effect.

The expression system is also intended to mean a cellular system, a cell-free system or a cell extract can be understood. However, the term “expression system” can also be used a system can be understood in which only the aspect of translation or the aspect the transcription is examined. In any case, in a way, phenotypically, ultimately the presence or absence of a glycosyltransferase turned off according to the method disclosed herein for the detection of glycosyltransferases is detected.

This is essentially the same in another aspect of the present Invention claimed method for identifying an activity, i. H. the enzymatic Compound influencing glycosyltransferase activity. It is also here preferably again an inhibitor that inhibits glycosyltransferase activity, i.e. H. reduced or completely prevented. The candidate connection is usually in one Reaction approach tested, which typically has another in addition to the glycosyltransferase Glycosyl donor and an immobilized glycosyl acceptor, which is preferably on one Cellulose membrane is immobilized. As with all of the invention disclosed herein Here too, the detection of one or the enzymatic glycosyltransferase Activity about 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 under the Influence of a glycosyltransferase arises, as easily through appropriate control approaches (Negative controls and positive controls) can be determined, as the person skilled in the art is common in this area.

All of the inventive methods disclosed herein have in common that they are due to the high Sensitivity, which not least allows a reduced sample volume, for the needs of High-throughput systems are suitable. Furthermore, the immobilization of the Glycosyl acceptor opens the possibility of site-specific reactions to prove what is particularly advantageous in the detection of the Transferase product, because here a handling of amounts of liquid, which is usually a limitation of high Throughput systems is avoided.

Finally, in another aspect, the object on which the invention is based solved by a kit. This kit can basically be used for or as part of everything here disclosed methods and uses are used. The kit is also based on the knowledge underlying the invention of the highly sensitive detection of Glycosyltransferase activities using an immobilized glycosyl acceptor. It is also within the scope of the present invention that such a kit instead of the already on the carrier, in particular a cellulose membrane, immobilized glycosyl acceptor too comprises a membrane and a glycosyl acceptor and optionally reagents for Immobilization of the glycosyl acceptor on or on (both terms are used synonymously here) the carrier included in the kit. To that extent, the term in connection with the Kit of a glycosyl acceptor immobilized on or on a support also for this purpose understood that this - typically - immediately before use from the Individual components is manufactured.

In addition to these components, the kit according to the invention can contain further components and Components include as provided in prior art kits. such Components can include, among other things, positive and negative controls, one or more Glycosyltransferases, one or more glycosyl donors, one or more Glycosyl acceptors as well as suitable buffers include.

The invention will now be explained in the following with reference to the figures and examples which result in further features, advantages and embodiments of the invention. there shows

Fig. 1 catalyzed by a α1,3 galactosyltransferase transfer of galactose;

Fig. 2 is catalyzed by an α1,3 / 4 fucosyltransferase transfer fucose;

Fig. 3 is catalyzed by a α2,3- (N) sialyltransferase transfer of sialic acid;

Fig. 4 is catalyzed by a sialyltransferase-α2,6- (N) Transfer of sialic acid;

FIG. 5 shows the immobilization of LacNAc-O- (CH2) ₈ COOH in an amino-functionalized cellulose membrane;

Fig. 6 shows the time-dependent enzymatic transfer in the presence of α1,3- galactosyltransferase;

Fig. 7 is an overview of different chemoselective reactions;

Fig. 8 is a graph showing a non-linear regression calculation for transferred galactose;

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

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

Fig. 11 shows the result of autoradiography of the α1,3 galactosyltransferase.

The various figures show the following in detail:

Fig. 1 The enzyme α1,3-galactosyltransferase catalyzes the transfer of Gal from UDP-Gal to the 3-hydroxyl function of the Gal residue from Galβ1 → 4GlcNAc-R to the compound Galα1 → 3 Galβ1 → 4GlcNAc-R.

Fig. 2 The enzyme α1,3 / 4-fucosyltransferase catalyzes the transfer of Fuc from GDP-Fuc to Gal residues in structures such as Galβ1 → 4GlcNAc-R (type II) and Galβ1 → 3GlcNAc-R (type I) to Le ^x and Le ^a structures.

Fig. 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 Galβ1 → 4GlcNAc-R (type II) or Galβ1 → 3GlcNAc-R (type I) containing structures.

Fig. 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 Galβ1 → 4GlcNAc-R or Galβ1 → 3GlcNAc-R.

• a) Fmoc β-Ala-OPfp, NMP;
• b) 10% Ac₂O in MeOH;
• c) Piperidin in DMF;
• d) LacNAc-O-(CH₂)₈COOH, PfpOH, DIC, NMP;
• e) 10% Ac₂O in MeOH.

Fig. 5 shows the derivatization of an amino-functionalized cellulose membrane (Glycinester-, photolinker membrane) with the acceptor LacNAc. a) The amino functions of the cellulose membrane are derivatized with the activated β-alanine building block "Fmoc-β-Ala-OPfp" b) After acetylation of the remaining amino groups with acetic anhydride, c) the Fmoc protecting group is split off with piperidine and d) the LacNAc building block Pfp ester immobilized. Finally, remaining amino functions are acetylated with acetic anhydride.

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

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

Fig. 6 shows the time-dependent enzymatic transfer of UDP-Gal (1.2 mM) in the presence of α1,3 galactosyltransferase (3.3 uU) 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 ).

Fig. 7 shows an overview of different chemoselective reactions according to the prior art: A) aldehydes (R ⁴ = H) or ketones (R ⁴ is not H), and amino-oxy compounds react to form 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 β-aminothiols react to β-mercaptoamides, F) mercaptans and maleimides react to succinimides. One of the reaction partners can be covalently linked to the glycosyl donor via R ¹ or R ⁴ , R ⁵ or R ⁶ or via a linker.

Fig. 8 shows a graphical representation of a non-linear regression calculation for galactose transferred in response to the UDP-Gal concentration for the α1,3- galactosyltransferase (see. Example 1 Determination of donor Km).

Fig. 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 0.78 fmol / min / mm ² , and the maximum transfer with 50 µmol / mm ² .

• 1. A: Kontrollmembran ohne Zugabe der Transferase
• 2. B: LacNAc gebunden an Glycinestermembran
• 3. C: zeitabhängiges Transferaseexperiment (2 h, 4 h, 24 h)

Fig. 10 shows the result of the transfer of fluoresceinyl-neuraminic acid by the α2,3- (N) -sialyltransferase (see Example 6). CMP-5-fluoresceinyl-neuraminic 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.

• 1. A: Control membrane without adding the transferase
• 2. B: LacNAc bound to glycine ester membrane
• 3. C: time-dependent transferase experiment (2 h, 4 h, 24 h)

Fig. 11 shows the Autoradiographieresultate the α1,3-galactosyltransferase after 6 days of exposure time (sec. Example 7).
ester 1: glycine ester membrane without immobilized LacNAc (negative control)
ester 2: LacNAc glycine ester membrane
photolinker 1: photolinker membrane without immobilized LacNAc (negative control)
photolinker 2: LacNAc photolinker membrane

The membrane strips were incubated for 24 h with ¹⁴ C-UDP-Gal in the presence of different α1,3-GalT concentrations: a) 132 µU b) 13.2 µU c) 1.32 µU.

A clear transfer can be achieved with an α1,3-GalT concentration of 132 µU in both LacNAc glycine ester membrane as well as the LacNAc photolinker membrane detected become. With an α1,3-GalT concentration of 13.2 µU the transfer can only be done with the Show LacNAc photolinker membrane (higher loading with LacNAc). The corresponding Negative controls (ester 1, photolinker 1) show negligible (α) or none (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
TLC: 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
Fmocβ-Ala-OPfp: N-β-Fmoc-β-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
h: hour, hour
HPLC: high-performance liquid chromatography
HTS: High throuput screening
L: liter
LacNAc: Galβ (1 → 4) GlcNAc
LacNAc-O- (CH ₂ ) ₈ COOH: Galβ (1 → 4) GlcNAcß (1 → 1) -8-octanoic acid
Le: Lewis
Le ^a : Galβ (1 → 3) [Fucα (1 → 4)] GlcNAc
Le ^x Galβ (1 → 4) [Fucα (1 → 3)] GlcNAc
Le ^y : Fucα (1 → 2) Galβ (1 → 4) [Fucα (1 → 3)] GlcNAc
M: molar
mAb: monoclonal antibody
Man: D-mannose
MeOH: methanol
MES: 2-morpholino-ethanesulfonic acid monohydrate
mL: milliliters
mM: millimolar
MOPS: 3-morpholino-propanesulfonic acid
mU: Milliunit
Neu5Ac, NeuAc: N-acetylneuraminic acid
NMP: 1-methyl-2-pyrrolidone
NMR: nuclear magnetic resonance
PBS: phosphate buffered saline
PfpOH: 2,3,4,5,6-pentafluorophenol
R: Molecular residue
SialT, ST: sialyltransferase
Sialyl-Le ^x , S-Le ^x : NeuAcα (2 → 3) Galβ (1 → 4) [Fucα (1 → 3)] GlcNAc
Tris: 2-amino-2-hydroxymethyl-1,3-propanediol
U: unit
UDP: uridine 5'-diphosphate
UV: ultraviolet
Xyl: D-xylose

For the examples described below, the following chemicals, Derivatization of the cellulose membranes and reaction conditions for the cleavage of the Connections used by the cellulose membrane.

chemicals

Cellulose membranes (filter paper 50) were manufactured by Whatman International Ltd. (Maidstone, England). 1-methyl-2-pyrrolidone (NMP), pentafluorophenol (PfpOH), 1,3-diisopropylcarbodiimide (DIC), and acetic anhydride are from Sigma-Aldrich (Germany). Methanol and N, N-dimethylformamide (DMF) come from Merck Eurolab GmbH (Germany). The photolinker 4- [4 (1-Fmoc-aminoethyl) -2-methoxy-5-nitrophenoxyl] butanoic acid, α1,3-fucosyltransferase (α (1 → 3) FT), and α2,6-sialyltransferase (α (2nd → 6) ST) were obtained from Calbiochem-Novabiochem (San Diego, USA). N ^α -Fmoc-β-Ala-OPfp is from Bachem Biochemica GmbH (Heidelberg, Germany). CMP-5-fluoresceinyl-neuraminic acid, tripotassium salt (CMP-5-fluoro-Neu5Ac) was obtained from Boehringer-Mannheim (Mannheim, Germany). UDP- [ ¹⁴ C] Gal, GDP- [ ¹⁴ 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 purchased from ICN (Costa Mesa, USA). BioMax MS Film is from Eastman Kodak Company (NY, USA).

Derivatization of the membranes

The photolinker derivatization of the cellulose membrane is carried out as already described [41]. N ^α -Fmoc-β-Ala-OPfp is coupled to the cellulose membrane in NMP ( 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, Lac-NAc-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 added by adding 100 µL Split diethylamine / water (50/50) into a cellulose strip / spot. After 1 h, 80 µL removed and the radioactivity determined in a scintillation counter.

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

EXAMPLE 1 α1,3-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), α1,3-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 size 2 glycine ester or photolinker membrane × 5 mm, 8-30 nmol) in a microcentrifuge tube to a final volume of 230 µL. Reaction mixtures are incubated for 24 h at room temperature. The membranes are then washed successively with a 1 M NaCl (15 mL, 50 ° C), buffer A (NaCl 8 g / L, KCl 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 combined with a mixture of radioactive ([ ¹⁴ C], 200,000 dpm) and non-radioactive UDP-Gal (1.2 mM) in the presence of α (1 → 3) GalT (3.3 µU) in one Microcentrifuge tube incubated. Six reaction vessels are prepared in this way and the reaction is started 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 scintillation 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 µmol / mm ² , or 6.25% of the total amount of LacNAc.

Determination of the donor km

Three pieces of membrane (2 mm dm) were incubated in the presence of UDP-Gal (200,000 dpm), α (1 → 3) GalT (3.3 µU), and alkaline phosphatase (0.1 U) in microcentrifuge tubes for 2 h. 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 α1,3-fucosyltransferase: radiochemical assay

In a similar manner (see Example 1), glycine esters and photolinker membranes 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), α1,3-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.

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 0.78 fmol / min / mm. The maximum transfer of GDP-Fuc was 1.1 µmol / mm ² , or 0.14% of the total amount of LacNAc ( Fig. 9).

EXAMPLE 3 α2,3- (N) -sialyltransferase: radiochemical assay

In a similar manner (see Example 1), 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), Incubated α2,3- (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-Neu5Ac 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 ² for the photolinker membrane.

EXAMPLE 4 α2,6- (N) -sialyltransferase: radiochemical assay

In a similar manner (cf. Example 1), 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) , α2,6- (N) -sialyltransferase (38 mU / mL, 5 µL) and assay buffer (250 mM sodium cacodylate, 0.5% Triton CF-54, pH = 6.0, 20 µL) in a final volume of 50 µL. The transfer rate of CMP-Neu5Ac 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 ² for the photolinker membrane.

EXAMPLE 5 α1,3-galactosyltransferase: mass spectroscopic assay

UDP-Gal (100 mM solution in water, 5 µL, 2 mmol), α1,3-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) was added to the membrane-bound acceptor (a strip of the glycine ester or photolinker membrane, 2 × 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 with 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 α2,3- (N) -sialyltransferase: fluorescence assay

CMP-5-fluoresceinyl-neuraminic acid (0.4 mM solution in water, 20 µL, 0.2 mM), α2,3- (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 nmol) 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 α1,3-galactosyltransferase: autoradiography 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), α1,3-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 ) 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 × 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) none Showed radioactivity. The amount of radioactivity transferred is proportional to Concentration of the enzyme used. The photolinker membranes generally showed higher ones Transfer rates.

EXAMPLE 8 α1,3-Fucosyltransferase: autoradiography assay

In a similar manner (cf. Example 7), glycine esters and photolinker membranes 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), α1,3-fucosyltransferase (164 mM, 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.

EXAMPLE 9 α2,3- (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), Incubate α2,3- (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

α2,6- (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 -Neu5Ac (300,000-400,000 dpm, in 1 µL water), α2 , 6- (N) -sialyltransferase (38 mM, 5 µL) and assay buffer (250 mM sodium cocodylate, 0.5% Triton CF-54, pH = 6, 20 µL) in a final volume of 50 µL.

Those disclosed in the above description, the claims and the drawings Features of the invention can be used both individually and in any combination for the Realization of the invention in its various embodiments may be essential.

Claims (36)

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 support, in particular a cellulose membrane, and
a transferase product is detected in the presence of an enzymatic glycosyltransferase activity. 2. The method according to claim 1, characterized in that the glycosyl donor in excess concentration is present, especially the excess at least twice Km value of the glycosyltransferase to be detected corresponds. 3. The method according to claim 1 or 2, characterized in that the glycosyl acceptor is selected from the group consisting of monosaccharides, oligosaccharides, in particular a part an oligosaccharide, polysaccharides and glycoconjugates such as glycoproteins, peptides and includes lipids. 4. The method according to claim 3, characterized in that it is the glycoconjugate is a lactosamine derivative, especially 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 (photolinkers) and carboxylic ester linkers, in particular glycine ester linker. 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 7, characterized in that the nucleotide sugar is selected is from the group that 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 is later labeled allows. 10. The method according to claim 9, characterized in that the marking is selected from the group consisting of fluorescent labels, radioactivity labels, Enzyme activity labels and chemiluminescent 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 glucuronyltransferases and glucosaminetransferases includes. 12. The method according to claim 11, characterized in that the galactosyltransferase a is 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 consisting of alpha-1,3 / 4-fucosyltransferases and alpha-1,2- Fucosyltransferases -V include. 14. The method according to claim 11, characterized in that the sialyltransferase is selected from the group consisting of alpha-2,3-sialyltransferases and alpha-2,6-sialyltransferases includes. 15. The method according to any one of claims 1 to 14, characterized in that the sample is selected from the group consisting of human, animal, plant and microbial samples includes. 16. The method according to claim 15, characterized in that the sample is selected from the group containing tissue, in particular tissue containing Golgi apparatus, and Includes body fluids. 17. The method according to claim 15, characterized in that the sample is selected from the group that purified cell lysates, tissue lysates, organ lysates, extracts, crude extracts Includes preparations and unpurified preparations. 18. The method according to claim 16, characterized in that the body fluid is selected from the group consisting of breast milk, urine, sputum, saliva, serum and Lymphatic fluid. 19. Method for the parallel detection of the enzymatic activity of Glycosyltransferase (s) in a large number of samples, characterized in that for each sample the Method according to one of claims 1 to 18 is carried out in parallel. 20. A method for determining the glycosyltransferase activity pattern of a sample, characterized in 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, and
optionally the transferase product is determined. 21. A method for determining the substrate specificity of a glycosyltransferase, characterized in that
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, and
optionally the transferase product is determined. 22. Use of a method according to one of the preceding claims for diagnosis and / or follow-up of diseases with an altered enzymatic Glycosyltransferase activity are linked. 23. Use according to claim 22, characterized in that the disease is selected is from the group, liver cirrhosis, immune deficiency diseases, lysosomal Includes storage diseases and tumor diseases. 24. Use according to claim 23, characterized in that the tumor disease is selected from the group consisting of human carcinomas, in particular melanomas, breasts, Colon, chorion, liver cell and bladder carcinomas, as well as animal carcinomas includes. 25. The method according to any one of the preceding claims, characterized in that the individual reaction steps 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 glycosyl transferase 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 demonstrated. 27. The method according to claim 26, characterized in that the connection Expression of glycosyltransferase inhibited. 28. The method according to claim 26 or 27, characterized in that the expression of the Is transcription. 29. The method according to claim 26 or 27, characterized in that the expression of the Translation is. 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 influence the activity of a glycosyltransferase in a reaction mixture, wherein the activity 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 reaction mixture, - and the presence or absence of a Transferase product is demonstrated. 31. The method according to claim 30, characterized in that the compound has the activity inhibits the glycosyltransferase. 32. The method according to any one of claims 26 to 31, characterized in that the Partial steps of the procedure in parallel with at least two different candidate connections be performed. 33. The method according to any one of claims 26 to 32, characterized in that the Candidate compound is included in a library of compounds, particularly one Library of low molecular weight compounds. 34. Kit for the detection of enzymatic glycosyltransferase activity comprising at least one immobilized on a support, in particular a cellulose membrane Glycosyl. 35. Kit according to claim 34, characterized in that the kit is a glycosyl donor includes. 36. Kit according to claim 34 or 35 comprising at least one further component which is selected from the group consisting of buffers, glycosyl donor (s), glycosyl acceptor (s) and Glycosyltransferase (s) includes.