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(12) United States Patent (10) Patent No.: US 9.410,182 B2 W (45) Date of Patent: Aug

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(12) United States Patent (10) Patent No.: US 9.410,182 B2 W (45) Date of Patent: Aug USOO941 0182B2 (12) United States Patent (10) Patent No.: US 9.410,182 B2 W (45) Date of Patent: Aug. 9, 2016 (54) PHOSPHATASE COUPLED OTHER PUBLICATIONS GLYCOSYLTRANSFERASE ASSAY Wu et al., R&D Systems Poster, “Universal Phosphatase-Coupled (75) Inventor: Zhengliang L. Wu, Edina, MN (US) Glycosyltransferase Assay”, Apr. 2010, 3 pages.* “Malachite Green Phosphate Detection Kit'. Research and Diagnos (73) Assignee: Bio-Techne Corporation, Minneapolis, tic Systems, Inc. Catalog No. DY996, Feb. 4, 2010, 6 pages.* MN (US) Zhu et al., Analytica Chimica Acta 636:105-110, 2009.* Motomizu et al., Analytica Chimica Acta 211:119-127, 1988.* *) NotOt1Ce: Subjubject to anyy d1Sclaimer,disclai theh term off thisthi Schachter et al., Methods Enzymol. 98.98-134, 1983.* patent is extended or adjusted under 35 Unverzagt et al., J. Am. Chem. Soc. 112:9308-9309, 1990.* U.S.C. 154(b) by 0 days. IUBMB Enzyme Nomenclature for EC 3.1.3.1, obtained from www. chem.cmul.ac.uk/iubmb? enzyme/EC3/1/3/5.html, last viewed on (21) Appl. No.: 13/699,175 Apr. 13, 2015, 1 page.* IUBMB Enzyme Nomenclature for EC 3.1.3.5, obtained from www. (22) PCT Filed: May 24, 2010 chem.cmul.ac.uk/iubmb? enzyme/EC3/1/3/5.html, last viewed on Apr. 13, 2015, 1 page.* (86). PCT No.: PCT/US2010/035938 Compain et al., Bio. Med. Chem. 9:3077-3092, 2001.* Lee et al., J. Biol. Chem. 277:49341-49351, 2002.* S371 (c)(1), Donovan R S et al., “A Solid-phase glycosyltransferase assay for (2), (4) Date: Nov. 20, 2012 high-throughput screening in drug discovery research.” Glycoconjugate Journal, vol. 16, No. 10, (Oct. 1, 1999), pp. 607-615. (87) PCT Pub. No.: WO2011/149448 Manu RM De Groeve et al., “Enzymatic production of O-d-galactose PCT Pub. Date: Dec. 1, 2011 1-phosphate by lactose phosphorolysis.” Biotechnology Letters, vol. 31, No. 12, (Jul 24, 2009), pp. 1873-1877. (65) Prior Publication Data Rowlands MG et al., “High-throughput screening assay for inhibi tors of heat-shock protein 90 ATPase activity.” Analytical Biochem US 2013/OO65262 A1 Mar. 14, 2013 istry, vol. 327, No. 2. (Apr. 15, 2004), pp. 176-183. PCT International Search Report dated Sep. 30, 2010 for PCT/ (51) Int. Cl. US2010/035938, from which the instant application is based, 3 pgs. CI2O 1/48 (2006.01) Written Opinion dated Sep. 30, 2010 for PCT/US2010/035938, from CI2O I/42 (2006.01) which the instant application is based, 5 pgs. (52) U.S. Cl. Wongkongkatep J et al., "Label-free, real-time glycosyltransferase CPC. CI2O 1/42 (2013.01): CI2O 1/48 (2013.01); assay based on a fluorescent artificial chemosensor.” Angewandte G0IN 2333/914 (2013.01); G0IN 2333/91091 Chemie, vol. 45, No. 4 (Jan. 16, 2006), pp. 665-668. (2013.01) (58) Field of Classification Search * cited by examiner CPC ............ C12Q 1/48; C12O 1/42; C12N 9/108; C12N 9/14: C12Y 306/01005; G01N Primary Examiner — David J Steadman 2333/91091: G01N 2333/914 USPC ...................................... 435/15, 18, 193, 195 (74) Attorney, Agent, or Firm — Fredrikson & Byron, P.A. See application file for complete search history. (57) ABSTRACT (56) References Cited A kit for detecting or measuring glycosyltransferase activity U.S. PATENT DOCUMENTS including a first reagent comprising a phosphatase and a 6,551,790 B2 * 4/2003 Trubetskoy et al. ............ 435/15 second reagent comprising a free phosphate detector, and 7,247,300 B1* 7/2007 Chen et al. ................... 424/94.6 method of detecting and measuring glycosyltransferase activ 7,521,250 B2 4/2009 Hamachi ity. A Sugar donor, an acceptor Substrate, a glycosyltrans 2008/0233592 A1 9/2008 Lowery ferase enzyme and a phosphatase are combined and the amount of free phosphate present in the product is measured FOREIGN PATENT DOCUMENTS and used to calculate the activity of the glycosyltransferase WO 941O340 A1 5, 1994 enzyme. WO WOOOf 42214 * 7/2OOO WO 02092845 A2 11/2002 WO WO 2008/036139 A2 * 3, 2008 5 Claims, 4 Drawing Sheets U.S. Patent Aug. 9, 2016 Sheet 3 of 4 US 9.410,182 B2 9eun61– 8800'0+X3.260'O=A Iz660=}} G?un61 U.S. Patent Aug. 9, 2016 Sheet 4 of 4 US 9.410,182 B2 & ( W d (in?uuroud) WS /eun61– O tae? (druu/oud) WS US 9,410,182 B2 1. 2 PHOSPHATASE COUPLED entail long assay times and high background readings, mak GLYCOSYLTRANSFERASE ASSAY ing the results less sensitive than desired. RELATED APPLICATIONS SUMMARY This application is a 35 U.S.C. 371 national stage filing Embodiments of the invention include assays and method from, and claims priority to, International Application No. of detecting and quantifying glycosyltransferase activity. In PCTUS2010/035938 filed May 24, 2010, the teachings of one embodiment, a kit for the detection of glycosyltransferase which are incorporated herein by reference. activity includes a first reagent comprising a phosphatase and 10 a second reagent comprising a free phosphate detector. The BACKGROUND phosphatase may be an apyrase such as ectonucleoside triph osphate diphosphohydrolase such as CD39L3 or a 5'-nucle Polysaccharides are extremely abundant and diverse, otidase such as CD73. In some embodiments, the phos including structures such as cellulose, chitin, starch, and gly phatase may be an alkaline phosphatase. The kit may further 15 include a phosphate free buffer. The kit may also include a cogen, to name just a few examples. Glycosylation is impor phosphate standard, such as KHPO. tant not only to the formation of carbohydrates, but also In some embodiments, the kit for detecting glycosyltrans glycoproteins, and glycolipids, cellular processes including ferase activity may include one or more nucleotides, such as protein folding, trafficking, recognition, and stability, cell UDP, GDP or CMP. The kit may include a sugar donor, such growth, migration, communication, and immune response. It as a Sugar nucleotide. The kit may also include a Sugar accep also plays a role in the virulence of many microorganisms. tor Substrate. In some embodiments, the kit may include a The formation of carbohydrate molecules in biological glycosyltransferase enzyme. systems depends upon glycosyltransferase enzymes, which Embodiments of the invention also include methods of perform the key function of transferring a monosaccharide or assaying the activity of a glycosyltransferase enzyme. The an oligosaccharide from a donor molecule to an acceptor. 25 method may include combining a Sugar donor, an acceptor Given the abundance and structural diversity of carbohy Substrate, the glycosyltransferase enzyme and a phosphatase drates, it is not Surprising that there are numerous glycosyl in a buffer Solution to produce a product, and measuring the transferase enzymes, each of which are specific to particular amount of free phosphate present in the product. The amount donor molecules and acceptor molecules. For example, it is of free phosphate present in the sample may then be used to estimated that the human genome encodes more than 200 30 calculate the activity of the glycosyltransferase enzyme. glycosyltransferase genes. In some embodiments, the Sugar donor, acceptor Substrate Glycosyltransferase enzymes add sugar groups not only to and glycosyltransferase may be combined first, and then the polysaccharides but also to proteins and lipids to form, gly phosphatase may be added. Alternatively, the Sugar donor, coproteins, and glycolipids. Most glycosyltransferases use acceptor Substrate, glycosyltransferase and phosphatase may activated nucleotide Sugars as donor molecules. These glyco 35 be combined at the same time in a coupling reaction. syltransferases are referred to as Leloir enzymes. A Leloir In some embodiments, the Sugar donor is a Sugar nucle glycosyltransferase transfers the Sugar group from a Sugar otide. In some embodiments, the Sugar nucleotide is Sugar nucleotide to a Substrate molecule (e.g., Sugar, protein or diphosphonucleotide and the phosphatase is an apyrase. Such lipid), generating a nucleotide leaving group. The other gly as an ectonucleoside triphosphate disphosphohydrolase. In cosyltransferases, referred to as non-Leloir enzymes, use 40 Some embodiments, the Sugar nucleotide is a Sugar mono other donors such as lipid phosphoSugars, resulting in leaving phosphonucleotide and the phosphatase is a monophospho groups such as lipid phosphates. nucleotidase, such as a 5'-nucleotidase. In other embodi Because of their key roles in development, cellular func ments, the Sugar donor is a lipid phospho-Sugar and the tions, and pathogenicity, it is desirable to monitor glycosyl phosphatase is an alkaline phosphatase. transferase activity. For example, glycosyltransferases may 45 In Some embodiments, the amount of free phosphate be ideal targets for drug therapies, to promote, inhibit, or present in the sample may be measured by applying a colo block their activities. A method of monitoring glycosyltrans rimetric assay to the sample. In some embodiments, the appli ferase activity would therefore be very useful in the develop cation of the colorimetric assay includes combining the prod ment of Such drugs, among other uses. uct with a first reagent comprising ammonium molybdate and Several methods have attempted to measure glycosyltrans 50 Sulfuric acid, then combining this with a second reagent com ferase activity, but each of these methods suffers from certain prising malachite green oxalate and polyvinyl alcohol, and difficulties which limit their usefulness. For example, one then measuring the light absorbance of the product of step bat traditional method of assaying glycosyltransferases uses 620 nm. The amount of free phosphate may then be calculated radioisotopes. However, besides involving the use of radio from the measured light absorbance. activity, this method requires the use of a specific labeled 55 In other embodiments, the invention includes a method of compound and separation of the donor from the acceptor, determining whether a test substance affects the activity of a which is a time consuming process.
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