USOO65343OOB1 (12) United States Patent (10) Patent No.: US 6,534,300 B1 Canfield (45) Date of Patent: Mar. 18, 2003

(54) METHODS FOR PRODUCING HIGHLY 5,089,392 A 2/1992 Miller et al...... 435/21 PHOSPHORYLATED LYSOSOMAL 5,126.247 A 6/1992 Palmer et al. ------435/25

HYDROLASES 2-Y/ / 2is a 2.0: whyll et al. his . . . . .almi......

(75) Inventor: wasO O M. Canfield, Oklahoma City, 5,202,2535,179,023 A 4/19931/1993 CalhounEsmon et et al. al...... 435/204.27435/320.1 5,205,917. A 4/1993 Klock, Jr...... 204/182.8 5,208,148 A 5/1993 Haugland et al...... 435/14 (73) Assignee: Genzyme Glycobiology Research 5,217.865 A E. yout------S. Institute, Inc., Oklahoma City, OK 24- a 2 US List continued on next pagpage. (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Kornfield, R, et al. (1976) Ann Rev. Biochem. 45, 217-237.* Altschuler, Y, et al. (2000) Mol. Biol. of the Cell 11, 819-831. 21 Ap1. No.: 09.1635,872 (21) ppl. No 1635, Alan D. Elbein et al., "Kifunensine, a Potent Inhibitor of the (22) Filed: Aug. 10, 2000 Glycoprotein Processing I'', The Journal of Biological Chemistry, vol. 265, No. 26, Issue of Sep. 15, pp. Related U.S. Application Data 15599-15605, 1990. (60) Provisional application No. 60/153.831, filed on Sep. 14, Kornfeld, R. et al. “Molecular Cloning and Functional 1999. Expression of Two Splice Forms of Human 51) Int. Cl...... C12N 9/14,s C12N 9/12 N-Acetvlglucosamine-1-phophodiestery1g pnop alpha-N-Acetvlp y (52)52) U.S. Cl...... 435/195./195; 435/435/194 9.lucosaminidase” J. Biol. Chem. 12, Nov. 1999. vol. 274, (58) Field of Search ...... 435/195, 194, No. 46, pp.32778-32785. 435/199 Cuozzo, J. W. et al. “Lysine is a Common Determinant for Mannose Phosphorylation of Lysosomal Proteins' J. Biol. (56) References Cited Chem. 20. May 1994. vol. 269, No. 20. pp. 14490–14496. Kornefeld, S. “Trafficking of Lysosomal in Nor U.S. PATENT DOCUMENTS mal and Disease States', J. Clin. Invest., vol. 77, Jan. 1986, 3,957,578 A 5/1976 Narita et al...... 195/11 pp. 1-6. 3.966,555 A 6/1976 Arnaud et al...... 195/66 R Kornefeld, S. “Lysosomal Targeting”, Biochemical E. A 2. and t l - - - e Society Transactions, Jubilee Lecture Delivered on Dec. 19, 2- : Y-2 a CCC C a - - - 4,156,013 A 5/1979 Bruinvels et al...... 424/319 1989, vol. 18. pp. 367-374. ---- 4,195,126 A 3/1980 Hall ...... 435/11 Sly, The Missing Link in Lysosomal Enzyme Targeting", 4,328.215 A 5/1982 Bueding ...... 424/181 The Journal of Clinical Investigation, vol. 105, No. 5, pp. 4,332,894 A 6/1982 Whistler ...... 435/99 563-564, Mar. 2000. s: A may s al t Raas-Rothschild et al., “Molecular Basis of Variant 4,431,7372 - - -a- A 2/1984 OlivieriOCal etC al.a ...... 435/208 Ext ye, Flyy Ely Melipid y) ss5. The 4,433,946 A 2/1984 Christianson et al...... 406/43 Ournal OI UIIn1cal InVeSugauon, Vol. , NO. , pp. 4,452,794. A 6/1984 Kort et al...... 424,240 673–681. Mar. 2000. 4,474,770 A 10/1984 Lorimeau et al. ... 424/1.83 Bao et al., “Bovine UDP-N-Acetylglucosamine: Lysosoma 4,492.761 A 1/1985 Durack ...... 436/519 l-Enzyme N-Acetylglucosamine-1-Phosphotransferase”, 4,496.722 A 1/1985 Gallop et al...... 544/69 The Journal of Biological Chemistry, vol. 271, No. 49, pp. 4,595,015 A 6/1986 Jansen et al...... 128/713 31446-31451 Dec. 6, 1996 4,615.884. A 10/1986 Harshman ...... 424/92 try . . . . . 4,639.420. A y S s ------i./7 Kornfield, Purification and Multimeric Structure of Bovine 4,659,817 A 4/1987 Gallop et al...... 540,541 N-Acetylglucosamine-1-Phosphodiester C-N-Acetylglu 4,749,570 A 6/1988 Poznansky ...... 424/94.3 cosaminidase”, The Journal of Biological Chemistry, vol. 4,798,169 A 1/1989 Rosen et al...... 119/3 273, No. 36, pp. 23203-23210, Sep. 4, 1998. 4,851,390 A 7/1989 Morishige ..... 514/44 4,868,042 A 9/1989 Neuwelt ...... 514/44 Primary Examiner-Charles L. Patterson, Jr. 4,975,441 A 12/1990 Gibson ...... 514/328 (74) Attorney, Agent, Or Firm-Oblon, Spivak, McClelland, 4,981,801 A 1/1991 Suzuki et al...... 435/296 Maier & Neustadt, PC 4,986.274 A 1/1991 Stephens ...... 128/660.07 9 - 8 Y- - 4,987.223 A 1/1991 Choay et al...... 536/17.7 (57) ABSTRACT 4997,760 A 3/1991 Hirabayashi et al. ... 435/227 5,001,072 A 3/1991 Olson ...... 436/500 The present invention provides highly phosphorylated lyso 5,015,470 A 5/1991 Gibson ...... 424/70 Somal , methods of modifying lysosomal hydro SE A 3. it." al - - - - - t5. lases with the lysosomal targeting pathway enzymes 5,061,0252Y- - - 2 A 10/1991 Mattesl, etJr. al.el al...... 435/172.3 Narbosphotransferase and/or phosphodiester 5075.231. A 12/1991 Moreau et al...... 435/198 ''N'S 5,077.200 A 12/1991 Habenstein ...... 435/14 5,082,778 A 1/1992 Overbeeke et al...... 435/172.3 36 Claims, 4 Drawing Sheets US 6,534,300 B1 Page 2

U.S. PATENT DOCUMENTS 5,691,181 A 11/1997 Lowe ...... 435/240.1 5,693,622 A 12/1997 Wolff et al...... 514/44 5.242,805 A 9/1993 Naleway et al...... 435/18 5,696,001 A 12/1997 Habenstein .... 436/518 SE A t E. Najima et al. s: 5,704,910 A 1/1998 Humes ...... 604/52 21 a-a-2 f Old ...... 422/ 5,707,865. A 1/1998 Kohn et al...... 435/325 5,296,365 A 3/1994. Overbeeke et al. . ... 435/208 5,310,646 A 5/1994 Whitle 435/4 5,716,614 A 2/1998 Katz et al...... 424/94.3 5316906 A 5/1994 Hau ideal------435/4 5,719,031 A 2/1998 Haugland et al. . ... 435/2.4 5,344,3522- - - 2 A 9/1994 Horne9. et al...... 445/24 5,721,967 A 2/1998 Kay et al...... 300/2 5,356.804 A 10/1994 Desnicket al. ... 435/268 5,7235852 1-2 A 3/1998 Baker et al...... 530/413 5.362.628 A 11/1994 Haugland ...... 435/18 5,728,381 A 3/1998 Wilson et al...... 424/94.6 5366,883 A 11/1994 Asada et al 435/202 RE35,770 E 4/1998 Lorimeau et al...... 514/56 2-Y/ / 2 - - - 5,736,360 A 4/1998 Gaulton et al. ... 435/69.1 5,382,524 A 1/1995 Desnicket al. ... 435/200 5,7419572 - / 2 A 4/1998 Deboer et al...... 800/2 5,401,650 A 3/1995 Desnicket al. ... 435/208 5,750,1722 as A 5/1998 Meade et al...... 426/580 5,405,751 A 4/1995 Roncarolo ...... 435/7.24 5,759.775.2 - Y-2 A 6/1998 Caras et al...... 435/6 5,420,112 A 5/1995 Lewis et al...... 514/12 2 - - 2 5,433,946 A 7/1995 Allen, Jr. et al. . . 424/94.3 5,773.2362 - 2 A 6/1998 Diwu et al...... 435/15 5.439.935 A 8/1995 Rawlings et al. ... 514/451 5,773.278 A 6/1998 Schuchman et al...... 435/358 5.443,986 A 8/1995 Haughland et al 435/4 5,792,647 A 8/1998 Roseman et al...... 435/252.3 2 : : - 5.798.239 A 8/1998 Wilson et al...... 435/183 5.449,604 A 9/1995 Schellenberg et al. ... 435/6 2 -- Y - 5,466.809 A 11/1995 Dime ...... 546/183 5,798.366 A 8/1998 Platt et al...... 514/315 2 : - / 2 5,798.448 A 8/1998 Caras et al...... 530/382.1 5,475,095 A 12/1995 Myerowitz ...... 536/23.1 2 -- Y - 5,491,075 A 2/1996. Desnicket al. ... 435/69.7 58O79432Y- - A 9/1998 Lee et al...... 526/238.2 5.494.810 A 2/1996 Barany et al. ... 435/91.52 5.830,711 A 11/1998 Barany et al. . ... 435/91.1 5,501,9572 - - -2 A 3/1996 Dennis et al. ... 435/15 5.830,8502Y-Y-2 A 11/1998 Gelb et al...... 514/2 5512471 A 4/1996 Smith ...... 435/208 5.830,916 A 11/1998 Hannum et al...... 514/625 2- --1-2 5.840,578 A 11/1998 Desnick ...... 435/325 5,534,615 A 7/1996 Baker et al...... 530/350 2Y- - - 2 5.5454O2 A 8/1996 Watkinson 424/94.63 5,849,885 A 12/1998 Nuyens et al...... 530/416 5,554.3662- - - 2 A 9/1996 Rawlings et - al.- - - ... 424/78.03 5851,7822Y-- a-2 A 12/1998 Hannun et al...... 435/7.71 5,565,362 A 10/1996 Rosen ...... 435/320.1 5,854.2072Y- 2 A 12/1998 Lee et al...... 514/2 5569.648 A 10/1996 Lewis et al. ... 514/12 5,858,351 A 1/1999 Podsakoff et al...... 424/93.2 5571,675. A 1/1996 Baker et a 435/6 5,858,744. A 1/1999 Baum et al...... 435/172.3 5,571,8932- as A 11/1996 Baker et al.- - - ... 530/350 5858.7552Y- a-2 A 1/1999 Lowe ...... 435/198 5576.424. A 11/1996 Mao et all 536/17.9 5,861,491 A 1/1999 Nuijens et al...... 530/417 2- . 12 - - - - - 5,871,946 A 2/1999 Lucas et al...... 435/18 5,578.479 A 11/1996 Laderman et al...... 435/202 5874,2972Y- -a- A 2/1999 Wu et al. 435/320.1 5,580,757 A 12/1996 Desnick et al...... 435/69.7 5870937 A 3/1999 R 435/325 5,583,160 A 12/1996 Igarashi et al. ... 514/669 2Y- - 2 oncarolo ...... 5,585,247 A 12/1996 Habenstein ...... 435/18 5,906,817 A 5/1999 Moullier et al...... 424/93.21 5,612.206 A 3/1997 Valerio et al...... 435/172.3 5,911,704 A 6/1999 Humes ...... 604/93 5621,106 A 4/1997 Dime ... 546/183 5,912,146 A 6/1999 Nishimura et al...... 435/91.1 5624.806 A 4/1997 Baker et all 435/7.1 5,914.231 A 6/1999 Hennink et al...... 435/6 Y/1 - 2 ------5916870 A 6/1999 Lee et al. ... 514/2 5,627,073. A 5/1997 Baker et al...... 435/331 2- - - 2 5627,171 A 5/1997 Park et al. . ... 514/114 5,916,911 A 6/1999 Shayman et al...... 514/428 5,633,2282Y-1 .. 2 A 5/1997 Lewis et al...... 514/12 5,917,122 A 6/1999 Byrne ...... 800/18 5,633.261. A 5/1997 Dime ...... 514/299 5,919,690 A 7/1999 Knap et al. .... 435/208 Y/ -- a 5919,913 A 7/1999 Nuyens et al...... 530/395 5,639.6O7. A 6/1997 Desnick et al...... 435/6 2- - - 2 y Y/ -- a 5928,928 A 7/1999 Aerts ...... 435/201 5,639,939 A 6/1997 McCune, III ...... 900/2 5929,0362 - 1-12 A 7/1999 McEver ...... 514/25 5,648.229 A 7/1997 Habenstein ...... 435/18 2 - 1 - 2 5,648,335 A 7/1997 Lewis et al...... 514/12 5,929,304. A 7/1999 Radin et al...... 800/288

5,658,567 A 8/1997 Calhoun et al...... 424/94.61 5,932.211 A 8/1999 Wilson et al...... 424/94.6 5,663,076 A 9/1997 Rostoker et al...... 438/14 5939.2792- - - 2 A 8/1999 Smith ...... 435/7.32 5,663.254 A 9/1997 Lee et al. 526/238.2 5,968.502 A 10/1999 Treco et al...... 424/93.21 5,665,366 A 9/1997 Rawlings et al...... 424/401 6,118,045 A 9/2000 Reuser et al...... 800/14 5,679,545 A 10/1997 Baker et al...... 435/69.1 5,686.240 A 11/1997 Schuchman et al...... 435/6 * cited by examiner U.S. Patent Mar. 18, 2003. Sheet 1 of 4 US 6,534,300 B1

A VO. 7

SubStrate y-Subunit Recognition DOmains

O-subunit- : Catalytis 3 ? Domains

6-subunit U.S. Patent Mar. 18, 2003 Sheet 2 of 4 US 6,534,300 B1

0IJI,KTORIJOHä NOIIWAILOVI E?IS

U.S. Patent US 6,534,300 B1

US 6,534,300 B1 1 2 METHODS FOR PRODUCING HIGHLY LySOSomal Targeting Pathway PHOSPHORYLATED LYSOSOMAL The lySOSomal targeting pathways have been Studied HYDROLASES extensively and the proceSS by which lySOSomal enzymes are Synthesized and transported to the has been This application claims priority to U.S. Provisional well described. Komfeld, S. (1986). “Trafficking of lysoso application No. 60/153,831 filed Sep. 14, 1999, and is mal enzymes in normal and disease States.' Journal of incorporated herein by reference. Clinical Investigation 77: 1-6 and Komfeld, S. (1990). “Lysosomal enzyme targeting.” Biochem. Soc. Trans. 18: BACKGROUND OF THE INVENTION 367-374. Generally, lysosomal enzymes are synthesized by 1O membrane-bound polySomes in the rough endoplastic 1. Field of the Invention reticulum (“RER”) along with secretory glycoproteins. In This invention relates generally to enzymes involved in the RER, lySOSomal enzymes acquire N-linked oligosaccha the lysosomal targeting pathway and particularly to isolated rides by the en-bloc transfer of a preformed oligosaccharide and purified GlcNAc-phosphotransferase and phosphodi from dolichol phosphate containing 2 N-acetylglucosamine, ester C-GlcNAcase, nucleic acids encoding the enzymes, 15 9-mannose and 3-glucose. Glycosylatedly SOSomal enzymes processes for production of recombinant GlcNAc are then transported to the Golgi apparatus along with phosphotransferase and phosphodiester C.-GlcNAcase, and Secretory proteins. In the cis-Golgi or intermediate compart the use of the enzymes for the preparation of highly phos ment lySOSomal enzymes are Specifically and uniquely phorylated lySOSomal enzymes that are useful for the treat modified by the transfer of GlcNAc-phosphate to specific ment of lySOSomal Storage diseases. mannoses. In a Second Step, the GlcNAc is removed thereby 2. Description of the Prior Art exposing the mannose 6-phosphate ("M6P) targeting deter minant. The lysosomal enzymes with the exposed M6P and LySOSomal Storage Diseases binds to M6P receptors in the trans-Golgi and is transported LySOSomes are organelles in eukaryotic cells that function to the endoSome and then to the lySOSome. In the lySOSome, 25 the phosphates are rapidly removed by lySOSomal phos in the degradation of macromolecules into component parts phatases and the mannoses are removed by lysosomal man that can be reused in biosynthetic pathways or discharged by nosidases (Einstein, R. and Gabel, C. A. (1991). “Cell- and the cell as waste. Normally, these macromolecules are ligand-specific deposphorylation of acid hydrolases: evi broken down by enzymes known as lySOSomal enzymes or dence that the mannose 6-phosphate is controlled by com lySOSomal hydrolases. However, when a lySOSomal enzyme partmentalization.” Journal of Cell Biology 112: 81-94). is not present in the lySOSome or does not function properly, The synthesis of lysosomal enzymes having exposed M6P the enzymes Specific macromolecular Substrate accumulates is catalyzed by two different enzymes, both of which are in the lySOSome as "Storage material” causing a variety of essential if the Synthesis is to occur. The first enzyme is diseases, collectively known as lysosomal storage diseases. UDP-N-acetylglucosamine : lysosomal enzyme LySOSomal Storage diseases can cause chronic illness and 35 N-Acetylglucosamine-1-phosphotransferase (“GlcNAc death in hundreds of individuals each year. There are phosphotransferase”) (E.C. 2.7.8.17). GlcNAc approximately 50 known lySOSomal Storage diseases, e.g., phosphotransferase cataly Ze S the transfer of Pompe Disease, , Fabry Disease, N-acetylglucosamine-1-phosphate from UDP-GlcNAc to Maroteaux-Lamy Syndrome ( VI), the 6 position of a C.1.2-linked mannoses on the lySOSonial (mucopolysaccharidosis IV), Hunter 40 enzyme. The recognition and addition of Syndrome (mucopolysaccharidosis II), Farber Disease, Acid N-acetylgluocosamine-1-phosphate to lysosomal hydrolases Lipase Deficiency, Krabbe Disease, and by GlcNAc-phosphotransferase is the critical and determin (mucopolysaccharidosis VII). In each of these diseases, ing Step in lysosomal targeting. The Second Step is catalyzed lySOSomes are unable to degrade a Specific compound or by N-acetylglucosamine-1-phosphodie Ster C.-N- group of compounds because the enzyme that catalyzes a 45 Acetylglucosaminidase ("phosphodiester C.-GlcNAcase”) Specific degradation reaction is missing from the lySOSome, (E.C. 3.1.4.45). Phosphodiester O-GlcNAcase catalyzes the is present in low concentrations in the lySOSome, or is removal of N-Acetylglucosamine from the GlcNAc present at Sufficient concentrations in the lySOSome but is not phosphate modified lySOSomal enzyme to generate a termi functioning properly. nal M6P on the lysosomal enzyme. Preliminary studies of LySOSomal Storage diseases have been Studied extensively 50 these enzymes have been conducted. Bao et al., in The and the enzymes (or lack thereof) responsible for particular Journal of Biological Chemistry, Vol. 271, Number 49, Issue diseases have been identified. Most of the diseases are of Dec. 6, 1996, pp. 31437-31445, relates to a method for caused by a deficiency of the appropriate enzyme in the the purification of bovine UDP-N-acetylglucosamine: Lyso lySOSome, often due to mutations or deletions in the Struc Somal enzyme N-Acetylglucosamine-1-phosphotransferase tural gene for the enzyme. For Some lysosomal Storage 55 and proposes a hypothetical Subunit Structure for the protein. diseases, the enzyme deficiency is caused by the inability of Bao et al., in The Journal of Biological Chemistry, Vol. 271, the cell to target and transport the enzymes to the lySOSome, Number 49, Issue of Dec. 6, 1996, pp. 31446-31451, relates e.g., I-cell disease and pseudo-Hurler polydystrophy. to the enzymatic characterization and identification of the LySOSomal Storage diseases have been Studied exten catalytic subunit for bovine UDP-N-acetylglucosamine: Sively and the enzymes (or lack thereof) responsible for 60 LySo Somal enzyme N-Acetylglucosamine-1- particular diseases have been identified (Scriver, Beaudet, phosphotransferase. Kornfeld et al., in The Journal of Bio Sly, and Vale, eds., The Metabolic Basis of Inherited logical Chemistry, Vol. 273, Number 36, Issue of Sep. 4, Disease, 6th Edition, 1989, Lysosomal Enzymes, Part 11, 1998, pp. 23203-23210, relates to the purification and Chapters 61-72, pp. 1565-1839). Within each disease, the multimeric Structure of bovine N-Acetylglucosamine-1- Severity and the age at which the disease presents may be a 65 phosphodiester C-N-Acetylglucosaminidase. However, the function of the amount of residual lySOSomal enzyme that proprietary monoclonal antibodies required to isolate these exists in the patient. proteins have not been made available to others and the US 6,534,300 B1 3 4 protein Sequences for the enzymes used in these preliminary It is another object of the present invention to provide Studies have not been disclosed. methods for producing recombinant GlcNAc Although the lySOSomal targeting pathway is known and phosphotransferase and recombinant phosphodiester the naturally occurring enzymes involved in the pathway O-GlcNAcase by culturing host cells that have been trans have been partially Studied, the enzymes responsible for fected or transformed with expression vectors having DNA adding M6P in the lySOSomal targeting pathway are difficult that encodes GlcNAc-phosphotransferase or phosphodiester to isolate and purify and are poorly understood. A better O-GlcNAcase. understanding of the lysosomal targeting pathway enzymes It is another object of the present invention to provide and the molecular basis for their action is needed to assist isolated and purified recombinant GlcNAc with the development of effective techniques for the utili phosphotransferase and recombinant phosphodiester Zation of these enzymes in methods for the treatment of O-GlcNAcase. lySOSomal Storage diseases, particularly in the area of tar It is another object of the present invention to provide geted enzyme replacement therapy. methods for the preparation of highly phosphorlyated lyso Somal enzymes that are useful for the treatment of lySOSomal Treatment of LySOSomal Storage Diseases 15 Lysosomal Storage diseases caused by the lack of Storage diseases. enzymes can in theory be treated using enzyme replacement It is a further object of the present invention to provide therapy, i.e., by administering isolated and purified enzymes highly phosphorlyated lysosomal hydrolases that are useful to the patient to treat the disease. However, to be effective, for the treatment of lysosomal Storage diseases. the lysosomal enzyme administered must be internalized by It is still another object of the present invention to provide the cell and transported to the lySOSome. Naturally occurring methods for the treatment of lySOSomal Storage diseases. enzymes and their recombinant equivalents, however, have It is still another object of the present invention to provide been of limited value in enzyme replacement therapy monoclonal antibodies capable of Selectively binding to because the purified or recombinantly SOSomal enzymes do bovine GlcNAc-phosphotransferase and to bovine phos not contain adequate amounts of exposed M6P, or contain 25 phodiester C.-GlcNAcase. undesirable oligosaccharides which mediates their destruc These and other objects are achieved by recovering iso tion. Without sufficient M6P, the administered lysosomal lated and purified biologically active GlcNAc enzyme cannot efficiently bind to M6P receptors and be phosphotransferase and phosphodiester C.-GlcNAcase and transported to the lySOSome. For example, human acid using the enzymes to obtain nucleic acid molecules that C-glucosidase purified from placenta contains oligomannose encode for the enzymes. The nucleic acid molecules coding oligosaccharides which are not phosphorylated (Mutsaers, J. for either enzyme are incorporated into expression vectors H. G. M., Van Halbeek, H., Vliegenthart, J. F. G., Tager, J. that are used to transfect host cells that express the enzyme. M., Reuser, A. J. J., Kroos, M., and Galjaard, H. (1987). The expressed enzyme is recovered and used to prepare “Determination of the structure of the carbohydrate chains highly phosphorylated lysosomal hydrolases useful for the of acid C-glucosidase from human placenta.” Biochimica et 35 treatment of lysosomal Storage diseases. In particular, the Biophysica Acta 911: 244-251), and this glycoform of the enzymes are used to produce highly phosphorylated enzyme is not efficiently internalized by cells (Reuser, A. J., lySOSomal hydrolases that can be effectively used in enzyme Kroos, M. A., Ponne, N.J., Wolterman, R. A., Loonen, M. replacement therapy procedures. C., Busch, H. F., Visser, W. J., and Bolhuis, P. A. (1984). LySOSomal hydrolases having high mannose Structures are “Uptake and stability of human and bovine acid alpha 40 treated with GlcNAc-phosphotransferase and phosphodi glucosidase in cultured fibroblasts and Skeletal muscle cells ester C.-GlcNAcase resulting in the production of from glycogenosis type II patients.” Experimental Cell asparagine-linked oligosaccharides that are highly modified Research 155:178-189). As a result of the inability to purify with mannose 6-phosphate (“M6P). The treated or Synthesize lySOSomal enzymes with the desired oligosac binds to M6P receptors on the cell membrane and is trans charide Structures, these enzyme preparations are ineffi 45 ciently targeted to affected cells and are of limited effec ported into the cell and delivered to the lysosome where it tiveness in the treatment of these diseases. There exists, can perform its normal or a desired finction. therefore, a need for enzymes that can be used in enzyme Other aspects and advantages of the present invention will replacement therapy procedures, particularly highly phos become apparent from the following more detailed descrip phorylated enzymes that will be efficiently internalized by 50 tion of the invention taken in conjunction with the accom the cell and transported to the lysosome. panying drawings. SUMMARY OF THE INVENTION BRIEF OF DESCRIPTION OF THE DRAWINGS It is, therefore, an object of the present invention to FIG. 1 shows a model of the Subunit structure of GlcNAc provide biologically active GlcNAc-phosphotransferase and 55 phosphotransferase. The enzyme is a complex of Six phosphodiester C.-GlcNAcase as isolated and purified polypeptides. The C- and B-Subunits are the product of a polypeptides. Single gene. Following translation, the C- and B-Subunits are It is another object of the present invention to provide separated by proteolytic cleavage between Lys' and nucleic acid molecule S encoding GlcNAc Asp''. The C-subunit is a type II membrane glycoprotein phosphotransferase and phosphodiester C.-GlcNAcase. 60 with a single amino terminal membrane Spanning domain. It is another object of the present invention to provide The B-Subunit is a type I membrane Spanning glycoprotein expression vectors having DNA that encodes GlcNAc with a Single carboxyl terminal membrane Spanning domain. phosphotransferase and phosphodiester C.-GlcNAcase. The Y-Subunit is the product of a Second gene. The Y-Subunit It is a further object of the present invention to provide is a Soluble protein with a cleaved signal peptide. The Cl-, 3-, host cells that have been transfected with expression vectors 65 and Y-Subunits are all tightly associated. having DNA that encodes GlcNAc-phosphotransferase or FIG. 2 shows a model of the subunit structure of phos phosphodiester O-GlcNAcase. phodiester C.-GlcNAcase. The enzyme is a tetramer com US 6,534,300 B1 S 6 posed of four identical Subunits arranged as two non or as a component of a larger nucleic acid construct that has covalently-associated dimers which are themselves been derived from DNA or RNA isolated at least once in disulfide-linked. The Single Subunit is a type I membrane Substantially pure form (i.e., free of contaminating endog protein containing a signal peptide, a pro region not present enous materials) and in a quantity or concentration enabling in the mature enzyme and a single carboxyl terminal mem identification, manipulation, and recovery of its component brane Spanning domain. nucleotide Sequences by Standard biochemical methods. FIG. 3 shows a diagram of recombinant glycoprotein Such Sequences are preferably provided in the form of an expression in CHO cells. In overexpressing CHO cells, the open reading frame uninterrupted by internal non-translated rh-GAA is processed along the pathways 1 and 2, depending Sequences, or introns that are typically present in eukaryotic on whether or not the enzyme is acted upon by GlcNAc genes. Sequences of non-translated DNA may be present 5' phosphotransferase (GnPT). Secreted GAA contains pre or 3' from an open reading frame where the same do not dominantly Sialylated biantenniary complex-type glycans interfere with manipulation or expression of the coding and is not a substrate for GlcNAc-phosphotransferase. In the region. presence of the C. 1,2-mannosidase inhibitors, The term “nucleic acid molecule' as used herein means 1-deoxymannojirimycin or kifunensine conversion of 15 RNA or DNA, including cDNA, single or double stranded, MAN9 to MAN5 structures is blocked, resulting in secretion and linear or covalently closed molecules. A nucleic acid of GAA-bearing MAN7-9 structures which can be modified molecule may also be genomic DNA corresponding to the with GlcNAc-phosphotransferase and phosphodiester entire gene or a Substantial portion therefor to fragments and O-GlcNAcase (UCE) generating phosphorylated Species derivatives thereof. The nucleotide Sequence may corre (pathway 3). spond to the naturally occurring nucleotide Sequence or may contain Single or multiple nucleotide Substitutions, deletions FIG. 4 shows transient expression analysis of various and/or additions including fragments thereof. All Such varia plasmid constucts of the C/B and Y Subunits of human tions in the nucleic acid molecule retain the ability to encode GlcNAc-phosphotransferase. Plasmids containing the C/B a biologically active enzyme when expressed in the appro and/or the Y subunits were transfected into 293T cells, the 25 priate host or an enzymatically active fragment thereof. The expressed protein was purified from the culture at 23, 44.5 nucleic acid molecule of the present invention may comprise and 70 hours after transfection and relative amounts of Solely the nucleotide Sequence encoding an enzyme or may expression were assessed by measuring phosphotransferase be part of a larger nucleic acid molecule that extends to the activity using methyl-O-D-mannoside and B-P UDP gene for the enzyme. The non-enzyme encoding Sequences GlcNAc as Substrates. in a larger nucleic acid molecule may include vector, DETAILED DESCRIPTION OF THE promoter, terminator, enhancer, replication, Signal INVENTION Sequences, or non-coding regions of the gene. The term “variant as used herein means a polypeptide The term “GlcNAc-phosphotransferase” as used herein Substantially homologous to a naturally occurring protein refers to enzymes that are capable of catalyzing the transfer 35 but which has an amino acid Sequence different from the of N-acetylglucosamine-1-phosphate from UDP-GlcNAc to naturally occurring protein (human, bovine, Ovine, porcine, the 6' position of C.1.2-linked mannoses on lySOSomal murine, equine, or other eukaryotic species) because of one enzymes. or more deletions, insertions, derivations, or Substitutions. The term “phosphodiester O-GlcNAcase” as used herein The variant amino acid sequence preferably is at least 50% refers to enzymes that are capable of catalyzing the removal 40 identical to a naturally occurring amino acid Sequence but is of N-Acetylglucosamine from GlcNAc-phosphate-mannose most preferably at least 70% identical. Variants may com diester modified lySOSomal enzymes to generate terminal prise conservatively Substituted Sequences wherein a given M6P. amino acid residue is replaced by a residue having Similar The terms “GlcNAc-phosphotransferase” and “phos physiochemical characteristics. Conservative Substitutions phodiester C.-GlcNAcase” as used herein refer to enzymes 45 are well known in the art and include Substitution of one obtained from any eukaryotic Species, particularly mamma aliphatic residue for another, Such as Ile, Val, Leu, or Ala for lian Species Such as bovine, porcine, murine, equine, and one another, or Substitutions of one polar residue for another, human, and from any Source whether natural, Synthetic, Such as between Lys and Arg, Glu and Asp, or Gln and ASn. Semi-synthetic, or recombinant. The terms encompass Conventional procedures and methods can be used for membrane-bound enzymes and Soluble or truncated 50 making and using Such variants. Other Such conservative enzymes having less than the complete amino acid Sequence Substitutions Such as Substitutions of entire regions having Similar hydrophobicity characteristics are well known. Natu and biologically active variants and gene products. rally occurring variants are also encompassed by the present The term “naturally occurring as used herein means an endogenous or exogenous protein isolated and purified from invention. Examples of Such variants are enzymes that result 55 from alternate MRNA splicing events or from proteolytic animal tissue or cells. cleavage of the enzyme that leave the enzyme biologically The term "isolated and purified” as used herein means a active and capable of performing its catalytic function. protein that is essentially free of association with other Alternate splicing of mRNA may yield a truncated but proteins or polypeptides, e.g., as a naturally occurring pro biologically active protein Such as a naturally occurring tein that has been Separated from cellular and other con 60 soluble form of the protein. Variations attributable to pro taminants by the use of antibodies or other methods or as a teolysis include differences in the N- or C-termini upon purification product of a recombinant host cell culture. expression in different types of host cells due to proteolytic The term “biologically active” as used herein means an removal of one or more terminal amino acids from the enzyme or protein having Structural, regulatory, or bio protein. chemical functions of a naturally occurring molecule. 65 The term “substantially the same” as used herein means The term “nucleotide Sequence' as used herein means a nucleic acid or amino acid Sequences having Sequence polynucleotide molecule in the form of a separate fragment variations that do not materially affect the nature of the US 6,534,300 B1 7 8 protein, i.e., the Structure and/or biological activity of the nucleic acid molecule S encoding GlcNAc protein. With particular reference to nucleic acid Sequences, phosphotransferase and its Subunits, expression vectors hav the term “substantially the same” is intended to refer to the ing a DNA that encodes GlcNAc-phosphotransferase, host coding region and to conserved Sequences governing expres cells that have been transfected or transformed with expres Sion and refers primarily to degenerate codons encoding the Sion vectors having DNA that encodes GlcNAc Same amino acid or alternate codons encoding conservative phosphotransferase, methods for producing recombinant substitute amino acids in the encoded polypeptide. With reference to amino acid Sequences, the term “Substantially GlcNAc-phosphotransferase by culturing host cells that the same' referS generally to conservative Substitutions have been transfected or transformed with expression vec and/or variations in regions of the polypeptide nor involved tors having DNA that encodes GlcNAc-phosphotransferase, in determination of Structure or function. isolated and purified recombinant GlcNAc The term "percent identity” as used herein means com phosphotransferase, and methods for using GlcNAc parisons among amino acid Sequences as defined in the phosphotransferase for the preparation of highly phospho UWGCG sequence analysis program available from the rylated lySOSomal enzymes that are useful for the treatment University of Wisconsin. (Devereaux et al., Nucl. Acids Res. of lySOSomal Storage diseases. 12:387-397 (1984)). 15 To obtain isolated and purified GlcNAc The term “highly phosphorylated lysosomal hydrolase' as phosphotransferase and its Subunits and the nucleic acid used to herein means a level of phosphorylation on a purified molecules encoding the enzyme according to the present lysosomal hydrolase which could not be obtained by only invention, bovine GlcNAc phosphotransferase was obtained isolating the hydrolase from a natural Source and without and analyzed as follows. Splenocytes from mice immunized Subsequent treatment with the GlcNAc-phosphotransferase with a partially purified preparation of bovine GlcNAc and phosphodiester-O-GlcNAcase. In particular, "highly phosphotransferase were fused with myeloma cells to gen phosphorylated lySOSomal hydrolase' means a lySOSomal erate a panel of hybridomas. Hybridomas Secreting mono hydrolase that contains from about 6% to about 100% clonal antibodies Specific for GlcNAc-phosphotransferase bis-phosphorylated oligosaccharides. were identified by immunocapture assay. In this assay, This invention is not limited to the particular 25 antibodies which could capture GlcNAc-phosphotransferase methodology, protocols, cell lines, vectors, and reagents from a crude Source were identified by assay of immuno described because these may vary. Further, the terminology precipitates with a specific GlcNAc-phosphotransferase used herein is for the purpose of describing particular embodiments only and is not intended to limit the Scope of enzymatic assay. Hybridomas were Subcloned twice, anti the present invention. AS used herein and in the appended body produced in ascites culture, coupled to a Solid Support claims, the singular forms “a,” “an,” and “the' include plural and evaluated for immunoaffinity chromatography. Mono reference unless the context clearly dictates otherwise, e.g., clonal PT18-Emphaze was found to allow a single step reference to “a host cell”) includes a plurality of such host purification of GlcNAc-phosphotransferase to homogeneity. cells. Bao, et al., The Journal of Biological Chemistry, Vol. 271, Because of the degeneracy of the genetic code, a multi Number 49, Issue of Dec. 6, 1996, pp. 31437-31445 relates tude of nucleotide Sequences encoding GlcNAc 35 to a method for the purification of bovine phosphotransferase, phosphodiester O-GlcNAcase, or other UDP-N-acetylglucosamine: Lysosomal-enzyme N-Acetyl Sequences referred to herein may be produced. Some of glucosamine-1-phosphotransferase and proposes a hypo these Sequences will be highly homologous and Some will be thetical Subunit Structure for the protein. Bao, et. al., The minimally homologous to the nucleotide Sequences of any Journal of Biological Chemistry, Vol. 271, Number 49, Issue known and naturally occurring gene. The present invention 40 of Dec. 6, 1996, pp. 31446-31451. Using this technique, the contemplates each and every possible variation of nucleotide enzyme was purified 488,000-fold in 29% yield. The eluted Sequence that could be made by Selecting combinations GlcNAc-phosphotransferase has a specific activity of >10, based on possible codon choices. These combinations are preferably >5x10, more preferably >12x10 pmol/h/mg made in accordance with the Standard triplet genetic code as and is apparently a homogenous, multi-Subunit enzyme applied to the nucleotide Sequence of naturally occurring 45 based on silver-stained SDS-PAGE. The monoclonal anti GlcNAc-phosphotransferase or phosphodie Ster body labeled PT18 was selected for use in further experi O-GlcNAcase, and all Such variations are to be considered as ments. A hybridoma secreting monoclonal antibody PT 18 being specifically disclosed. was deposited with the American Type Culture Collection, Unless defined otherwise, all technical and Scientific 10801 University Blvd., Manassas, Va. 20110 on Aug. 29, terms and any acronyms used herein have the same mean ings as commonly understood by one of ordinary skill in the 50 2000 and assigned ATCC Accession No. PTA 2432. art in the field of the invention. Although any methods and GlcNAc-phosphotransferase was determined to be a com materials similar or equivalent to those described herein can plex of Six polypeptides with a Subunit Structure C.B.Y. be used in the practice of the present invention, the preferred FIG. 1 shows a model of the Subunit structure obtained from methods, devices, and materials are described herein. quantitative amino acid Sequencing, immunoprecipitation All patents and publications mentioned herein are incor 55 with subunit-specific monoclonal antibodies, SDS-PAGE, porated herein by reference to the extent allowed by law for and cDNA sequences. The evidence for the model is sum the purpose of describing and disclosing the proteins, marized below. The molecular mass of the complex esti enzymes, vectors, host cells, and methodologies reported mated by gel filtration is 570,000 Daltons. The 166,000 therein that might be used with the present invention. Dalton C-Subunit is found as a disulfide-linked homodimer. However, nothing herein is to be construed as an admission 60 Likewise, the 51,000 Dalton Y-subunit is found as a that the invention is not entitled to antedate Such disclosure disulfide-linked homodimer. Because both the C- and by virtue of prior invention. Y-Subunits are found in disulfide-linked homodimers, each THE INVENTION molecule must contain at least one O- and one Y homodimer. Although the 56,000 Dalton B-subunit is not found in a GlcNAc-phosphotransferase 65 disulfide-linked homodimer, two independent lines of evi In one aspect, the present invention provides isolated and dence Strongly Suggest each complex contains two purified biologically active GlcNAc-phosphotransferase, B-Subunits as well. First, quantitative aminoterminal US 6,534,300 B1 9 10 Sequencing demonstrates a 1:1 molar ratio between the B expressed sequence tag (“EST) files, full-length human and Y-Subunits. Secondly, Since the C- and B-Subunits are cDNAS encoding each Subunit were cloned and Sequenced. encoded by a single cDNA and divided by proteolytic The nucleotide Sequence for the human C/B-Subunit pre processing, two B-Subunits are produced for each C-Subunit cursor cDNA is shown in nucleotides 165-3932 of SEO ID dimer. The predicted mass of the complex based on the NO:4; the nucleotide sequence for the C-subunit is shown in composition CBY is 546,000 Daltons (2x166,000+2x56, nucleotides 165-2948 of SEQ ID NO:4; the nucleotide 000+2x51,000) in excellent agreement with the mass esti Sequence for the C-Subunit is shown in nucleotides mated by gel filtration. 2949–3932 of SEQ ID NO:4; and the nucleotide sequence GlcNAc-phosphotransferase was purified using an assay for the Y-subunit is shown in nucleotides 96-941 of SEQ ID for the transfer of GlcNAc-1-Phosphate to the synthetic NO:5. The nucleotide sequence for the Y-subunit signal acceptor C.-methylmannoside. However, the natural accep peptide is shown in nucleotides 24-95 of SEQ ID NO:5. tors for GlcNAc-phosphotransferase are the high mannose For each subunit a N-terminal peptide and two internal oligosaccharides of lySOSomal hydrolases. To evaluate the peptide Sequences have been identified in the respective ability of the purified GlcNAc-phosphotransferase to utilize cDNA sequence. Although the protein Sequence data is from glycoproteins as acceptors, the transfer of GlcNAc-1-P to the bovine protein and the cDNA sequences are human, the the lySOSomal enzymes uteroferrin and cathepsin D, the 15 Sequences are highly homologous (identities: C-Subunit nonlySOSomal glycoprotein RNASe B, and the lySOSomal 43/50; B-subunit 64/64; Y-subunit 30/32), confirming the hydrolase f3- (which is trafficked by a cloned cDNAS represent the human homologs of the bovine M6P independent pathway), were investigated. Both utero GlcNAc-phosphotransferase subunits. The C- and ferrin and cathepsin D are effectively utilized as acceptors by B-subunits were found to be encoded by a single cDNA purified GlcNAc-phosphotransferase with KS below 20 whose gene is on chromosome 12. The Y-Subunit is the tim. In contrast, neither RNASe B nor B-glucocerebrosidase product of a Second gene located on chromosome 16. The is an effective acceptor. C/B-Subunits precursor gene has been cloned and Sequenced. The ineffectiveness of RNASe B, which contains a single The gene spans ~80 kb and contains 21 exons. The Y-subunit high mannose oligosaccharide, as an acceptor is especially gene has also been identified in data reported from a genome notable Since the K, was not reached at the Solubility limit 25 Sequencing effort. The Y-Subunit gene is arranged as 11 of the protein (at 600 um). This data clearly demonstrates the exons Spanning 12 kb of genomic DNA. Specific phosphorylation of LySOSomal hydrolaseS previ Using the human cDNAS, the homologous murine cDNAS ously observed with crude preparations (Waheed, Pohlmann for the Cl-, 3- and Y-Subunits were isolated and Sequenced A., R., et al. (1982). “Deficiency of UDP-N- using Standard techniques. The murine C.- B-Subunit precur acetylglucosa mine : l y So Somal enzyme Sor cDNA is shown in SEO ID NO:16. The deduced amino N-Acetylglucosamine-lphosphotransferase in organs of acid sequence for the murine O-subunit is shown in SEQ ID I-Cell patients.” Biochemical and Biophysical Research NO:15 and the B-subunit in SEQ ID NO:8. Communications 105(3): 1052–10580 is a property of the The mouse Y-Subunit cDNA was isolated from a mouse GlcNAc-phosphotransferase itself. liver library in 2 Zap II using the Y-human Y-subunit cl)NA 35 as a probe. The human Y-subunit cl)NA was random The C-subunit was identified as containing the UDP hexamer-labeled with P-dCTP and used to screen a mouse GlcNAc Since this Subunit was specifically liver cDNA library in Zap II. The probe hybridized to three photoaffinity-labeled with B-'P-5-azido-UDP-Glc. of 500,000 plaques screened. Each was subcloned to The amino-terminal and internal (tryptic) protein homogeneity, the insert excised, cloned into puC19, and Sequence data was obtained for each Subunit. N-terminal 40 Sequenced using Standard methods Sarnbrook, J., Fritsch E. Sequence was obtained from each Subunit as follows. Indi F., et al. (1989). Molecular Cloning. A Laboratory Manual. vidual subunits of GlcNAc-phosphotransferase were Cold Spring Harbor, Cold Spring Harbor Laboratory Press. resolved by polyacrylamide gel electrophoresis in the pres The mouse Y-subunit cl)NA sequence is shown in SEQ ID ence of sodium dodecyl sulfate before and after disulfide NO:10 and the deduced amino acid sequence for the mouse bond reduction. Subunits were then transferred to a PVDF 45 Y-subunit is shown in SEQ ID NO:9. membrane by electroblotting, identified by Coomassie blue Comparison of the deduced amino acid Sequences of the Staining, excised, and Subjected to N-terminal Sequencing. human and mouse Cl-, 3-, and Y-Subunits demonstrates that To obtain internal Sequence, GlcNAc-phosphotransferase the proteins are highly homologous with about an 80 percent was denatured, reduced, and alkylated, and individual Sub identity. units were resolved by gel filtration chromatography. ISO 50 lated Subunits were then digested with trypsin and the tryptic To confirm that these enzymes were substantially the peptides fractionated by reverse phase HPLC. Peaks which Same between Species, a partial homologous rat cDNA for appeared to contain only a single peptide were analyzed for the C- and B-Subunits was isolated and Sequenced using purity by MALDI and subjected to N-terminal amino acid standard techniques. The partial rat C.- and B-subunit cl)NA Sequencing. is shown in SEQ ID NO:12. The deduced amino acid 55 sequence corresponding to the cDNA is shown in SEQ ID The amino acid Sequence for the human C-Subunit is NO:11. Further, a partial homologous Drosophila cDNA for shown in amino acids 1-928 of SEQ ID NO:1; the human the C- and B-Subunits was isolated and Sequenced using B-subunit in amino acids 1-328 of SEQ ID NO:2; and the Standard techniques. The partial Drosophila C- and human Y-subunit in amino acids 25-305 of SEQ ID NO:3. |B-subunit cDNA is shown in SEQ ID NO:17. The deduced The Y-Subunit has a signal Sequence shown in amino acids 60 amino acid Sequence corresponding to the cDNA is shown 1-24 of SEO ID NO:3. in SEQ ID NO:13. Comparisons of the deduced amino acid Comparison with the databases using the blast algorithms Sequences of the partial human, rat, and Drosophila C- and demonstrate these proteins have not been previously B-Subunits show that the proteins are highly homologous. described although Several EST Sequences of the corre sponding cDNAS are present. 65 Phosphodiester C.-GlcNAcase Using these peptide Sequences and a combination of In another aspect, the present invention provides isolated library screening, RACE, PCR and Blast searching of and purified biologically active phosphodie Ster US 6,534,300 B1 11 12 O-GlcNAcase, nucleic acid molecules encoding phosphodi the capture assay, each hybridoma was Subcloned twice and ester C.-GlcNAcase, expression vectors having a DNA that antibody prepared by ascites culture. Monoclonals UC2 and encodes phosphodiester C.-GlcNAcase, host cells that have UC3 were found to be low affinity antibodies. UC1, a high been transfected or transformed with expression vectors affinity IgG monoclonal antibody, was prepared by ascites having DNA that encodes phosphodiester C.-GlcNAcase, culture and immobilized on Emphaze for purification of methods for producing recombinant phosphodiester phosphodiester O-GlcNAcase. The monoclonal antibody O-GlcNAcase by culturing host cells that have been trans labeled UC1 was selected for use in further experiments. A fected or transformed with expression vectors having DNA hybridoma Secreting monoclonal antibody UC1 was depos that encodes phosphodiester C.-GlcNAcase, isolated and ited with the American Type Culture Collection, 10801 purified recombinant phosphodiester O-GlcNAcase, and University Blvd., Manassas, Va. 20110 on Aug. 29, 2000 and methods for using phosphodiester O-GlcNAcase for the assigned ATCC Accession No. PTA 2431. preparation of highly phosphorylated lySOSomal enzymes To purify phosphodiester C.-GlcNAcase, a solubilized that are useful for the treatment of lysosomal Storage dis membrane fraction was prepared from bovine liver. Phos phodiester O-GlcNAcase was absorbed to monoclonal anti CSCS. body UC1 coupled to Emphaze resin by incubation over To obtain isolated and purified phosphodiester 15 O-GlcNAcase and the nucleic acid molecules encoding the night with gentle rotation. The UC1-Emphaze was then enzyme according to the present invention, bovine phos packed in a column, washed Sequentially with EDTA and phodiester a GlcNAcase was obtained and analyzed as NaHCO at pH 7.0, then phosphodiester O-GlcNAcase was follows. Mice were immunized with a partially purified eluted with NaHCO at pH 10. Fractions containing phos preparation of phosphodiester C.-GlcNAcase and a func phodiester C.-GlcNAcase at specific activities >50,000 u/mg tional Screening Strategy was utilized to identify and isolate were pooled and adjusted to pH 8.0 with /5th volume of 1 a monoclonal antibody Specific for phosphodiester M Tris HCI, pH 7.4. Following chromatography on UCI O-GlcNAcase. Immunogen was prepared by partially puri EmphaZe the phosphodiester O-GlcNAcase was purified fying phosphodiester C.-GlcNAcase -6000-fold from a 92,500-fold in 32% yield. bovine pancreas membrane pellet using chromatography on 25 The phosphodiester C.-GlcNAcase from UC1-Emphaze DEAE-Sepharose, iminodiacetic acid Sepharose, and Super was concentrated and chromatographed on Superose 6. ose 6. Two BALB/c mice were each injected intraperito Phosphodiester C.-GlcNAcase eluted early in the chromato neally with 5 lug partially purified phosphodiester gram as a Symmetric activity peak with a coincident protein O-GlcNAcase emulsified in Freunds complete adjuvant. On peak. Following chromatography on Superose 6, the enzyme day 28, the mice were boosted intraperitoneally with 5 lug was purified -715,000-fold in 24% yield. The purified phosphodiester C.-GlcNAcase emulsified in Freunds incom enzyme catalyzed the cleavage of 472 umols/hr/mg H plete adjuvant. On day 42 the mice were bled and an GlcNAc-1-phosphomannose-O-methyl, corresponding to a phosphodiester O-GlcNAcase specific immune response specific activity of 472,000 units/mg. was documented by “capture assay.” To perform the capture The purified phosphodiester O-GlcNAcase was subjected assay, Serum (5 ul) was incubated overnight with 1.2 units 35 to SDS-PAGE and protein was detected by silver staining partially purified phosphodiester C.-GlcNAcase. Mouse anti (Blum, H., Beier H., et al. (1987). “Improved silver staining body was then captured on rabbit antimouse IgG bound to of plant proteins, RNA and DNA in polyacrylamide gels.” protein A-UltralinkTM resin. Following extensive washing, Electrophoresis: 93–99). A diffuse band was observed with bound phosphodiester C.-GlcNAcase was determined in the a molecular mass of approximately 70 kDa whose intensity Ultralink pellet by assay of cleavage of H-GlcNAc-1- 40 varies with the measured phosphodiester C.-GlcNAcase phosphomannose C.-methyl. activity. The diffuse appearance of the band Suggests the Following a Second intravenous boost with phosphodi protein may be heavily glycosylated. A faint band with a ester C.-GlcNAcase, the Spleen was removed and Spleno molecular mass of ~150,000, which does not correlate with cytes fused with SP2/0 myeloma cells according to our activity, was also present. modifications (Bag, M., Booth J. L., et al. (1996). “Bovine 45 A model for the subunit structure of phosphodiester UDP-N-acetylglucosamine : lysosomal enzyme O-GlcNAcase was determined by gel filtration chromatog N-acetylglucosamine-1-phosphotransferase. I. Purification raphy and SDS-PAGE with and without disulfide bond and subunit structure.” Journal of Biological Chemistry 271: reduction. The mass by gel filtration is about 300,000. 31437-31445) of standard techniques; Harlow, E. and Lane, SDS-PAGE without disulfide bond reduction is -140,000. D. (1988). Antibodies: a laboratory manual, Cold Spring 50 Following disulfide bond reduction, the apparent mass is Harbor Laboratory). The fusion was plated in eight 96-well 70,000. Together these data show phosphodiester plates in media Supplemented with recombinant human IL-6 O-GlcNAcase is a tetramer composed of disulfide linked (Bazin, R. and Lemieux, R. (1989). “Increased proportion of homodimers. FIG. 2 shows a model of the Subunit structure B cell hybridomas Secreting monoclonal antibodies of of phosphodiester O-GlcNAcase. desired Specificity in cultures containing macrophage 55 The amino terminal amino acid Sequence of affinity derived hybridoma growth factor (IL-6).” Journal of Immu purified, homogeneous bovine phospho die Ster nological Methods 116: 245–249) and grown until hybrido O-GlcNAcase was determined using Standard methods mas were just visible. Forty-eight pools of 16-wells were (Matsudaira, P., Ed. (1993). A Practical Guide to Protein constructed and a SSayed for antiphosphodie Ster and Peptide Purification for MicroSequencing. San Diego, O-GlcNAcase activity using the capture assay. Four pools 60 Academic Press, Inc.). The pure enzyme was also Subjected were positive. Subpools of 4-wells were then constructed to trypsin digestion and HPLC to generate two internal from the wells present in the positive 16-well pools. Three tryptic peptides which were Sequenced. The amino acid of the four 16-well pools contained a single 4-well pool with Sequences of these three peptides are: anti-phosphodiester C.-GlcNAcase activity. The 4 single Peptide 1–Amino Terminal DXTRVHAGRLEHESWP Wells making up the 4-well pools were then assayed indi 65 PAAQTAGAHRPSVRTFV (SEQ ID NO:23): vidually identifying the well containing the anti Peptide 2-Tryptic RDGTLVTGYLSEEEVLDTEN phosphodiester O-GlcNAcase Secreting hybridomas. Using (SEQ ID NO:24); and US 6,534,300 B1 13 14 Peptide 3–Tryptic GINLWEMAEFLLK (SEQ ID where identity exceeds 90%. The murine gene for NO:25). phosphodiester C.-GlcNAcase is shown in SEQ ID NO:14. The protein, nucleotide, and EST data bases were The human phosphodiester C.-GlcNAcase gene has been Searched for Sequences that matched these peptide identified by database Searching. The Sequence was deter Sequences and Several human and mouse ESTs were found mined during the Sequencing of clone 165E7 from chromo that had the Sequence of the third peptide at their amino some 16.13.3, GenBank AC007011. 1, gi4371266. termini. Three human infant brain EST clones and one Interestingly, the phosphodiester C-GlcNAcase gene was mouse embryo clone were obtained from ATCC and not identified by the SCAN program used to annotate the Sequenced. The three human clones were all identical except Sequence. for total length at their 3' ends and virtually identical to the Because of the degeneracy of the genetic code, a DNA mouse clone, except that the mouse EST contained a 102 bp sequence may vary from that shown in SEQ ID NO:4, SEQ region that was absent from all three human brain ESTs. An ID NO:5, and SEQ ID NO:7 and still encode a GlcNAc EcoRI-Hind III fragment of about 700 bp was excised from phosphotransferase and a phosphodiester O-GlcNAcase the human cDNA clone (ATCC #367524) and used to probe enzyme having the amino acid Sequence shown in SEQ ID a human liver cDNA library directionally cloned in Trip1EX 15 NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:6. vector (Clontech). Of the positive clones isolated from the Such variant DNA sequences may result from silent library and converted to plasmids (pTrip1EX), the largest mutations, e.g., occurring during PCR amplification, or may (2200 bp) was represented by clone 6.5 which was used for be the product of deliberate mutagenesis of a native the rest of the analysis. Sequence. The invention, therefore, provides equivalent iso The CDNA clone has been completely sequenced on both lated DNA sequences encoding biologically active GlcNAc Strands and is a novel Sequence that predicts a mature protein phosphotransferase and phosphodiester C.-GlcNAcase of about 50 kDa which is in agreement with the size of the Selected from: (a) the coding region of a native mammalian deglycosylated mature bovine liver phosphodiester GlcNAc-phosphotransferase gene and phosphodiester O-GlcNAcase. O-GlcNAcase gene; (b) cDNA comprising the nucleotide There is a unique BamH I site at base #512 and a unique 25 sequence presented in SEQ ID NO:4, SEQ ID NO:5, and Hind ID site at base # 1581. All three bovine peptide SEQ ID NO:7; (c) DNA capable of hybridization to the Sequences (peptides 1, 2, and 3) were found. Although the native mammalian GlcNAc-phosphotransferase gene and sequences of peptides 2 and 3 in the human are 100% phosphodiester O-GlcNAcase gene under moderately Strin identical to the bovine Sequences, the amino-terminal pep gent conditions and which encodes biologically active tide in humans is only 67% identical to the bovine sequence. GlcNAc-phosphotransferase and phosphodie Ster The human liver clone contains the 102 base pair insert that O-GlcNAcase; and (d) DNA which is degenerate as a result has the characteristics of an alternatively spliced Segment of the genetic code to a DNA defined in (a), (b), or (c) and that was missing in the human brain EST. The hydrophilicity which encodes biologically active GlcNAc plot indicates the presence of a hydrophobic membrane phosphotransferase and phosphodiester C.-GlcNAcase. Spanning region from amino acids 448 to 474 and another 35 GlcNAc-phosphotransferase and phosphodie Ster hydrophobic region from amino acid 8 to 24 which fits the O-GlcNAcase proteins encoded by such DNA equivalent motif for a signal Sequence and there is a likely signal Sequences are encompassed by the invention. Sequence cleavage Site between G24 and G25. There are Six Those Sequences which hybridize under Stringent condi Asn-X-Ser/Thr potential N-linked glycosylation sites, one of tions and encode biologically functional GlcNAc which is within the 102 bp insert. All of these sites are amino 40 phosphotransferase and phosphodiester C.-GlcNAcase are terminal of the putative trans-membrane region. These fea preferably at least 50-100% homologous, which includes tures indicate that the phosphodiester O-GlcNAcase is a type 55, 60, 65,70, 75.75, 80, 85,90, 95, 99% and all values and I membrane Spanning glycoprotein with the amino terminus Subranges therebetween. Homology may be determined with in the lumen of the Golgi and the carboxyl terminus in the the software UWCG as described above. Stringent hybrid cytosol. This orientation is different from that of other 45 ization conditions are known in the art and are meant to glycosyltransferases and glycosidases involved in glycopro include those conditions which allow hybridization to those tein processing, which to date have been shown to be type Sequences with a specific homology to the target Sequence. II membrane Spanning proteins. An example of Such Stringent conditions are hybridization at The amino acid Sequence for the phosphodiester 65 C. in a standard hybridization buffer and Subsequent O-GlcNAcase monomer is shown in amino acids 50-515 of 50 washing in 0.2x concentrate SSC and 0.1% SDS at 42–65° SEQ ID NO:6. The signal peptide is shown in amino acids C., preferably 60° C. This and other hybridization conditions 1-24 of SEQ ID NO:6 and the pro segment is shown in are disclosed in Sambrook, J., Fritsch E. F., et al. (1989). amino acids 25-49 of SEO ID NO:6. The human cDNA was Molecular Cloning. A Laboratory Manual. Cold Spring cloned using the techniques described above. The nucleotide Harbor, Cold Spring Harbor Laboratory Press. Alternatively, Sequence for the monomer that associates to form the 55 the temperature for hybridization conditions may vary phosphodiester C.-GlcNAcase tetramer is shown in nucle dependent on the percent GC content and the length of the otides 151–1548 of SEQID NO:7. The nucleotide sequence nucleotide Sequence, concentration of Salt in the hybridiza for the signal sequence is shown in nucleotides 1-72 of SEQ tion buffer and thus the hybridization conditions may be ID NO:7. The nucleotide sequence for the propeptide is calculated by means known in the art. shown in nucleotides 73-150 of SEO ID NO:7. 60 Recombinant Expression for GlcNAc-phosphotransferase The murine cDNA for phosphodiester O-GlcNAcase is and Phosphodiester C.-GlcNAcase Isolated and purified shown in SEQID NO:18. The deduced amino acid sequence recombinant GlcNAc-phosphotransferase and phosphodi for the murine phosphodiester C.-GlcNAcase is shown in ester C.-GlcNAcase enzymes are provided according to the SEQ ID NO:19. Comparison of the deduced amino acid present invention by incorporating the DNA corresponding Sequences of the human and mouse enzymes demonstrates 65 to the desired protein into expression vectors and expressing that the proteins are highly homologous with about an 80 the DNA in a suitable host cell to produce the desired percent identity. This is especially true in the region of the protein. US 6,534,300 B1 15 16 Expression Vectors Expression vectors for use in prokaryotic host cells gen Recombinant expression vectors containing a nucleic acid erally comprise one or more phenotypic Selectable marker Sequence encoding the enzymes can be prepared using well genes. A phenotypic Selectable marker gene is, for example, known techniques. The expression vectors include a DNA a gene encoding a protein that conferS antibiotic resistance Sequence operably linked to Suitable transcriptional or trans 5 or that Supplies an autotrophic requirement. Examples of lational regulatory nucleotide Sequences Such as those useful expression vectors for prokaryotic host cells include derived from mammalian, microbial, Viral, or insect genes. those derived from commercially available plasmids Such as Examples of regulatory Sequences include transcriptional the cloning vector pBR322 (ATCC 37017). pBR322 con promoters, operators, enhancers, mRNA ribosomal binding tains genes for amplicillin and tetracycline resistance and Sites, and appropriate Sequences which control transcription thus provides simple means for identifying transformed and translation initiation and termination. Nucleotide cells. To construct an expression vector using p3R322, an Sequences are “operably linked' when the regulatory appropriate promoter and a DNA sequence are inserted into Sequence functionally relates to the DNA sequence for the the pBR322 vector. appropriate enzyme. Thus, a promoter nucleotide Sequence Other commercially available vectors include, for is operably linked to a GlcNAc-phosphotransferase or phos 15 example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, phodiester a GlcNAcase DNA sequence if the promoter Sweden) and pGEM1 (Promega Biotec, Madison, Wis., nucleotide Sequence controls the transcription of the appro USA). priate DNA sequence. Promoter Sequences commonly used for recombinant The ability to replicate in the desired host cells, usually prokaryotic host cell expression vectors include B-lactamase conferred by an origin of replication and a Selection gene by (penicillinase), lactose promoter System (Chang et al., which transformants are identified, may additionally be Nature 275:615, (1978); and Goeddelet al., Nature 281:544, incorporated into the expression vector. (1979)), tryptophan (trp) promoter system (Goeddel et al., In addition, Sequences encoding appropriate Signal pep Nucl. Acids Res. 8:4057, (1980)), and tac promoter tides that are not naturally associated with GlcNAc (Maniatis, Molecular Cloning: A Laboratory Manual, Cold phosphotransferase or phosphodiester C.-GlcNAcase can be 25 Spring Harbor Laboratory, p. 412 (1982)). incorporated into expression vectors. For example, a DNA Sequence for a signal peptide (Secretory leader) may be Yeasts useful as host cells in the present invention include fused in-frame to the enzyme Sequence So that the enzyme those from the genus Saccharomyces, Pichia, K. Actino is initially translated as a fusion protein comprising the mycetes and Kluyveromyces. Yeast vectors will often con Signal peptide. A signal peptide that is functional in the tain an origin of replication Sequence from a 2u yeast intended host cells enhances extracellular Secretion of the plasmid, an autonomously replicating sequence (ARS), a appropriate polypeptide. The Signal peptide may be cleaved promoter region, Sequences for polyadenylation, Sequences from the polypeptide upon secretion of enzyme from the for transcription termination, and a Selectable marker gene. cell. Suitable promoter Sequences for yeast vectors include, 35 among others, promoters for metallothione in, Host Cells 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. Suitable host cells for expression of GlcNAc 255.2073, (1980)) or other glycolytic enzymes (Holland et phosphotransferase and phosphodiester at O-GlcNAcase al., Biochem. 17:4900, (1978)) such as enolase, include prokaryotes, yeast, archae, and other eukaryotic glyceraldehyde-3-phosphate dehydrogenase, hexokinase, cells. Appropriate cloning and expression vectors for use 40 pyruvatee decarboxylase, phosphofructokinase, glucose-6- with bacterial, fingal, yeast, and mammalian cellular hosts phosphate , 3-phosphoglycerate mutase, pyruvate are well known in the art, e.g., Pouwels et al. Cloning kinase, triosephosphate isomerase, phosphoglucose Vectors: A Laboratory Manual, Elsevier, New York (1985). isomerase, and glucokinase. Other Suitable vectors and The vector may be a plasmid vector, a Single or double promoters for use in yeast expression are further described Stranded phage vector, or a Single or double-Stranded RNA 45 in Fleer et al., Gene, 107:285-195 (1991). Other suitable or DNA viral vector. Such vectors may be introduced into promoters and vectors for yeast and yeast transformation cells as polynucleotides, preferably DNA, by well known protocols are well known in the art. techniques for introducing DNA and RNA into cells. The Yeast transformation protocols are known to those of skill vectors, in the case of phage and Viral vectors also may be in the art. One such protocol is described by Hinnen et al., and preferably are introduced into cells as packaged or 50 Proceedings of the National Academy of Sciences USA, encapsulated virus by well known techniques for infection 75:1929 (1978). The Hinnen protocol selects for Trp.sup.+ and transduction. Viral vectors may be replication compe transformants in a Selective medium, wherein the Selective tent or replication defective. In the latter case viral propa medium consists of 0.67% yeast nitrogen base, 0.5% gation generally will occur only in complementing host casamino acids, 2% glucose, 10 ug/ml adenine, and 20 tug/ml cells. Cell-free translation Systems could also be employed 55 uracil. to produce the enzymes using RNAS derived from the Mammalian or insect host cell culture Systems well present DNA constructs. known in the art could also be employed to express recom Prokaryotes useful as host cells in the present invention binant GlcNAc-phosphotransferase or phosphodiester include gram negative or gram positive organisms. Such as E. O-GlcNAcase polypeptides, e.g., Baculovirus Systems for coli or Bacili. In a prokaryotic host cell, a polypeptide may 60 production of heterologous proteins in insect cells (Luckow include a N-terminal methionine residue to facilitate expres and Summers, Bio/Technology 6:47 (1988)) or Chinese Sion of the recombinant polypeptide in the prokaryotic host hamster ovary (CHO) cells for mammalian expression may cell. The N-terminal Met may be cleaved from the expressed be used. Transcriptional and translational control Sequences recombinant GlcNAc-phosphotransferase or phosphodiester for mammalian host cell expression vectors may be excised O-GlcNAcase polypeptide. Promoter Sequences commonly 65 from Viral genomes. Commonly used promoter Sequences used for recombinant prokaryotic host cell expression vec and enhancer Sequences are derived from Polyoma virus, tors include B-lactamase and the lactose promoter System. Adenovirus 2, Simian Virus 40 (SV40), and human cytome US 6,534,300 B1 17 18 galovirus. DNA sequences derived from the SV40 viral include various insoluble matrices comprising Sulfopropyl genome may be used to provide other genetic elements for or carboxymethyl groups. Further, one or more reversed expression of a structural gene Sequence in a mammalian phase high performance liquid chromatography (RP-HPLC) host cell, e.g., SV40 origin, early and late promoter, steps employing hydrophobic RP-HPLC media (e.g., silica enhancer, Splice, and polyadenylation sites. Viral early and gel having pendant methyl or other aliphatic groups) can be late promoters are particularly useful because both are easily employed to further purify the enzyme. Some or all of the obtained from a viral genome as a fragment which may also foregoing purification Steps, in various combinations, are contain a viral origin of replication. Exemplary expression well known in the art and can be employed to provide an vectors for use in mammalian host cells are well known in isolated and purified recombinant protein. the art. Recombinant protein produced in bacterial culture is The enzymes of the present invention may, when usually isolated by initial disruption of the host cells, beneficial, be expressed as a fusion protein that has the centrifugation, extraction from cell pellets if an insoluble enzyme attached to a fusion Segment. The fusion Segment polypeptide, or from the Supernatant fluid if a Soluble often aids in protein purification, e.g., by permitting the polypeptide, followed by one or more concentration, Salting fusion protein to be isolated and purified by affinity chro 15 out, ion eXchange, affinity purification, or size exclusion matography. Fusion proteins can be produced by culturing a chromatography steps. Finally, RP-HPLC can be employed recombinant cell transformed with a fusion nucleic acid for final purification steps. Microbial cells can be disrupted Sequence that encodes a protein including the fusion Seg by any convenient method, including freeze-thaw cycling, ment attached to either the carboxyl and/or amino terminal Sonication, mechanical disruption, or use of cell lysing end of the enzyme. Preferred fusion Segments include, but agents. are not limited to, glutathione - S-, B-galactosidase, a poly-histidine Segment capable of binding Preparation of Highly Phosphorylated Lysosomal to a divalent metal ion, and maltose binding protein. In Enzymes addition, the HPC-4 epitope purification system may be In another aspect, the present invention provides highly employed to facilitate purification of the enzymes of the 25 phosphorylated lySOSomal hydrolases and methods for the present invention. The HPC-4 system is described in U.S. preparation of Such hydrolases. The highly phosphorylated Pat. No. 5,202,253, the relevant disclosure of which is herein lySOSomal hydrolases can be used in clinical applications for incorporated by reference. the treatment of lysosomal Storage diseases. The method comprises obtaining lySOSomal hydrolases Expression by Gene Activation Technology having asparagine-linked oligosaccharides with high man In addition to expression Strategies involving transfection nose Structures and modifying the C.1.2-linked or other outer of a cloned cDNA sequence, the endogenous GlcNAc mannoses by the addition of M6P in vitro to produce a phophotransfease and phosphodiester C.-GlcNAcase genes hydrolase that can be used for the treatment of lysosomal can be expressed by altering the promoter. 35 storage diseases because it binds to cell membrane M6P Methods of producing the enzymes of the present inven receptors and is readily taken into the cell and into the tion can also be accomplished according to the methods of lySOSome. Typically, the high mannose Structures consist of protein production as described in U.S. Pat. No. 5,968,502, from Six to nine molecules of mannose and two molecules the relevant disclosure of which is herein incorporated by of N-acetylglucosamine (GlcNAc). In the preferred reference, using the Sequence S for GlcNAc 40 embodiment, the high mannose Structure is a characteristic phosphotransferase and phosphodiester C.-GlcNAcase as MAN7(DD) isomer structure consisting of seven mol described herein. ecules of man nose and two molecules of N-acetylglucosamine (GlcNAc). Expression and Recovery Highly phosphorylated LySOSomal hydrolases are pro According to the present invention, isolated and purified 45 duced by treating the high mannose hydrolases with GlcNAc-phosphotransferase or phosphodie Ster GlcNAc-phosphotransferase which catalyzes the transfer of O-GlcNAcase enzymes may be produced by the recombi N-acetylglucosamine-1-phosphate from UDP-GlcNAc to nant expression Systems described above. The method com the 6' position of C.1.2-linked or other Outer mannoses on the prises culturing a host cell transformed with an expression hydrolase. This GlcNAc-phosphotransferase modified vector comprising a DNA sequence that encodes the enzyme 50 hydrolase is then treated with phosphodiester O-GlcNAcase under conditions Sufficient to promote expression of the which catalyzes the removal of N-Acetylglucosamine to enzyme. The enzyme is then recovered from culture medium generate terminal M6P on the hydrolase. or cell extracts, depending upon the expression System In one embodiment of the invention, the GlcNAc employed. AS is known to the Skilled artisan, procedures for phosphotransferase treated hydrolase may be isolated and purifying a recombinant protein will vary according to Such 55 Stored without any Subsequent treatment. Subsequently, the factors as the type of host cells employed and whether or not GlcNAc-phosphotransferase treated hydrolase may be the recombinant protein is Secreted into the culture medium. modified further by treating the hydrolase with a phosphodi When expression Systems that Secrete the recombinant pro ester O-GlcNAcase. tein are employed, the culture medium first may be concen Surprisingly, it has been found that the hydrolases con trated. Following the concentration Step, the concentrate can 60 taining M6P generated by this method are highly phospho be applied to a purification matrix Such as a gel filtration rylated when compared to naturally occurring or known medium. Alternatively, an anion exchange resin can be recombinant hydrolases. The highly phosphorylated lySOSo employed, e.g., a matrix or Substrate having pendant diethy mal hydrolases of the present invention contain from about laminoethyl (DEAE) groups. The matrices can be 6% to about 100% bis-phosphorylated oligosaccharides acrylamide, agarose, dextran, cellulose, or other types com 65 compared to less that about 5% bis-phosphorylated oligosac monly employed in protein purification. Also, a cation charides on known naturally occurring or recombinant eXchange Step can be employed. Suitable cation exchangers hydrolases. US 6,534,300 B1 19 20 These highly phosphorylated hydrolases have a higher mination of the Structure of the carbohydrate chains of acid affinity for the M6P receptor and are therefore more effi C-glucosidase from human placenta.” Biochimica et Bio ciently taken into the cell by plasma membrane receptors. physica Acta 911: 244-251). The arrangement of the phos (Reuser, A.J., Kroos, M.A., Ponne, N.J., Wolterman, R. A., phates as either bis- or monophosphorylated oligosaccha Loonen, M. C., Busch, H. F., Visser, W. J., and Bolhuis, P. rides has not been determined, but less than 1% of the A. (1984). “Uptake and stability of human and bovine acid oligosaccharides contain any M6P. alpha-glucosidase in cultured fibroblasts and Skeletal muscle The highly phosphorylated hydrolases of the present cells from glycogenosis type II patients.” Experimental Cell invention are useful in enzyme replacement therapy proce Research 155:178–189). dures because they are more readily taken into the cell and The high-affinity ligand for the cation-independent M6P the lysosome. (Reuser, A. J., Kroos, M. A., Ponne, N.J., receptor is an oligosaccharide containing two M6P groups Wolterman, R. A., Loonen, M. C., Busch, H. F., Visser, W. (i.e., a bis-phosphorylated oligosaccharide). Since a bispho J. and Bolhuis, P. A. (1984). “Uptake and stability of human sphorylated oligosaccharides binds with an affinity 3500 and bovine acid alpha-glucosidase in cultured fibroblasts fold higher than a monophosphorylated oligosaccharides, and skeletal muscle cells from glycogenosis type II Virtually all the high-affinity binding of a lySOSomal enzyme 15 patients.” Experimental Cell Research 155: 178-189). to the M6P receptor will result from the content of bis Any lysosomal enzyme that uses the M6P transport sys phosphorylated oligosaccharides (Tong, P. Y., Gregory, W., tem can be treated according to the method of the present and Komfeld, S. (1989)). “Ligand interactions of the cation invention. Examples include O-glucosidase (Pompe independent mannose 6-phosphate receptor. The Stoichiom Disease), C.-L-iduronidase (Hurler Syndrome), etry of mannose 6-phosphate binding.” Journal of Biologi O-galactosidase A (Fabry Disease), arylsulfatase cal Chemistry 264: 7962–7969). It is therefore appropriate (Maroteaux-Lamy Syndrome), N-acetylgalactosamine-6- to use the content of bis-phosphorylated oligosaccharides to Sulfatase or B-galactosidase (Morquio Syndrome), iduronate compare the binding potential of different preparations of 2-sulfatase (), ceramidase (Farber lySOSomal enzymes. Disease), galacto cerebrosidase (Krabbe Disease), The extent of mannose 6-phosphate modification of two 25 f-glucuronidase (Sly Syndrome), Heparan N-sulfatase different lysosomal enzymes has been published. The oli (Sanfilippo A), N-Acetyl-O-glucosaminidase (Sanfilippo B), gosaccharide composition of human C-galactosidase A Acetyl Co A-C.-glucosaminide N-acetyl transferase, Secreted from Chinese hamster ovary cells has been pub N-acetyl-glucosamine-6 Sulfatase (Sanfilippo D), Galactose lished (Matsuura, F., Ohta, M., Ioannou, Y.A., and Desnick, 6-sulfatase (Morquio A), Arylsulfatase A, B, and C R. I. (1998). “Human alpha-galactosidase A: characteriza (Multiple Sulfatase Deficiency), Arylsulfatase A Cerebro tion of the N-linked oligosaccharides on the intracellular and side (Metachromatic Leukodystrophy), Ganglioside Secreted glycoforms overexpressed by Chinese hamster (Mucolipidosis IV), Acid f-galactosidase G. Galglioside ovary cells.” Glycobiology 8(4): 329-39). Of all oligosac (G. Ganglio Sidosis), Acid B-galactosidase charides on C-gal A released by hydrazinolysis, only 5.2% (Galactosialidosis), A (Tay-Sachs and were bis-phosphorylated. Zhao et al. partially characterized 35 Variants), Hexosaminidase B (Sandhoff), C.- the oligosaccharide Structures on recombinant human (FucSidosis), Cl-N-Acetyl galactosaminidase (Schindler C-iduronidase secreted by CHO cells (Zhao, K.W., Faull, K. Disease), Glycoprotein (Sialidosis), Aspar F., Kakkis, E. D., and Neufeld, E. F. (1997). “Carbohydrate tylglucosamine amidase (Aspartylglucosaminuria), Acid Structures of recombinant human alpha-L-iduronidase Lipase (Wolman Disease), Acid Ceramidase (Farber secreted by Chinese hamster ovary cells.” J Biol Chem 40 Lipogranulomatosis), Lysosomal Sphingomyelinase and 272(36): 22758-65) and demonstrated a minority of the other Sphingomyelinase (Nieman-Pick). oligosaccharides were bisphosphorylated. The qualitative Methods for treating any particularly SOSomal hydrolase techniques utilized precluded the determination of the frac with the enzymes of the present invention are within the skill tion of oligosaccharides phosphorylated. of the artisan. Generally, the lySOSomal hydrolase at a The production and Secretion of human acid 45 concentration of about 10 mg/ml and GlcNAc C-glucosidase by CHO cells has been reported (Van Hove, phosphotransferase at a concentration of about 100,000 J. L., Yang, H. W., Wu, J. Y., Brady, R. O., and Chen, Y.T. units/mL are incubated at about 37 C. for 2 hours in the (1996). “High level production of recombinant human lyso presence of a buffer that maintains the pH at about 6-7 and Somal acid alpha-glucosidase in Chinese hamster ovary cells any Stabilizers or coenzymes required to facilitate the reac which targets to heart muscle and corrects glycogen accu 50 tion. Then, phosphodiester C.-GlcNAcase is added to the mulation in fibroblasts from patients with Pompe disease.” system to a concentration of about 1000 units/mL and the Proceedings of the National Academy of Sciences USA, system is allowed to incubate for about 2 more hours. The 93(1): 6570). The carbohydrate structures of this preparation modified lysosomal enzyme having highly phosphorylated were not characterized in this publication. However, this oligosaccharides is then recovered by conventional means. preparation was obtained and analyzed. The results, given in 55 In a preferred embodiment, the lysosomal hydrolase at 10 the examples below, showed that less than 1% of the mg/ml is incubated in 50 mm Tris-HCI, pH 6.7, 5 mM oligosaccharides contained any M6P and bis MgCl, 5 mM MnC1, 2 mM UDP-GlcNAc with GlcNAc phosphorylated oligosaccharides were not detectable. phosphotransferase at 100,000 units/mL at 37 C. for 2 Together, these data show that known preparations of hours. Phosphodiester C.-GlcNAcase, 1000 units/mL, is then recombinantlySOSomal enzymes contain no more than 5.2% 60 added and the incubation continued for another 2 hours. The phosphorylated oligosaccharides. It appears that the prepa modified enzyme is then repurified by chromatography on ration of more highly phosphorylated lySOSomal enzymes is Q-Sepharose and step elution with NaCl. unlikely to be achieved with known techniques. Naturally occurring human acid C-glucosidase purified from human Methods for Obtaining High Mannose Lysosomal placenta contains very low levels of M6P (Mutsaers, I. H. G. 65 Hydrolases M., Van Halbeek, H., Vliegenthart, J. F. G., Tager, J. M., High mannose lysosomal hydrolases for treatment Reuser, A. J. J., Kroos, M., and Galjaard, H. (1987). “Deter according to the present invention can be obtained from any US 6,534,300 B1 21 22 convenient Source, e.g., by isolating and purifying naturally phosphorylated, 62% being bis-phosphorylated, and 12% occurring enzymes or by recombinant techniques for the monophosphorylated. Since each molecule of rh-GAA con production of proteins. High mannose lySOSomal hydrolases tains 7 N-linked oligosaccharides, 100% of the rh-GAA can be prepared by expressing the DNA encoding a particu molecules are likely to contain the mannose-phosphate lar hydrolase in any host cell System that generates a modification. oligosaccharide modified protein having high mannose Any alpha 1.2-mannosidase inhibitor can function in the Structures, e.g., yeast cells, insect cells, other eukaryotic present invention. Preferably, the inhibitor is selected from cells, transformed Chinese Hamster Ovary (CHO) host cells, the group consisting of deoxymannojirimycin (dMM), or other mammalian cells. kifunensine, D-Mannonolactam amidraZone, and N-butyl In one embodiment, high mannose lySOSomal hydrolases deoxymannojirimycin. Most preferably the inhibitor is are produced using mutant yeast that are capable of express deoxymannojimycin. ing peptides having high mannose Structures. These yeast include the mutant S. cervesiae A ochl, Amnnl (Nakanishi Treatment of LySOSomal Storage Diseases Shindo, Y., Nakayama, K. I., Tanaka, A., Toda, Y. and In a further aspect, the present invention provides a Jigami, Y. (1993). “Structure of the N-linked oligosaccha method for the treatment of lySOSomal Storage diseases by rides that show the complete loSS of C-1,6-polymannose 15 administering a disease treating amount of the highly phos outer chain from ochl, ochl minnl, and ochl minnl alg3 phorylated lySOSomal hydrolases of the present invention to mutants of Saccharomyces cerevisiae.” Journal of Biologi a patient Suffering from the corresponding lySOSomal Storage cal Chemistry 268: 26338–26345). disease. While dosages may vary depending on the disease Preferably, high mannose lySOSomal hydrolases are pro and the patient, the enzyme is generally administered to the duced using over-expressing transformed insect, CHO, or patient in amounts of from about 0.1 to about 1000 milli other mammalian cells that are cultured in the presence of grams per 50 kg of patient per month, preferably from about certain inhibitors. Normally, cells expressing lySOSomal 1 to about 500 milligrams per 50 kg of patient per month. hydrolases Secrete acid O-glucosidase that contains pre The highly phosphorylated enzymes of the present invention dominantly Sialylated biantenniary complex type glycans 25 are more efficiently taken into the cell and the lySOSome than that do not serve as a substrate for GlcNAc the naturally occurring or leSS phosphorylated enzymes and phosphotransferase and therefore cannot be modified to use are therefore effective for the treatment of the disease. the M6P receptor. Within each disease, the severity and the age at which the According to the present invention, a new method has disease presents may be a function of the amount of residual been discovered for manipulating transformed cells contain lySOSomal enzyme that exists in the patient. AS Such, the ing DNA that expresses a recombinant hydrolase So that the present method of treating lysosomal Storage diseases cells Secrete high mannose hydrolases that can be modified includes providing the highly phosphorylated lySOSomal according to the above method. In this method, transformed hydrolases at any or all Stages of disease progression. cells are cultured in the presence of C.1.2-mannosidase The lysosomal enzyme is administered by any convenient inhibitors and the high mannose recombinant hydrolases are 35 means. For example, the enzyme can be administered in the recovered from the culture medium. Inhibiting alpha 1,2- form of a pharmaceutical composition containing the mannosidase prevents the enzyme from trimming mannoses enzyme and any pharmaceutically acceptable carriers or by and forces the cells to Secrete glycoproteins having the high means of a delivery System Such as a liposome or a con mannose Structure. High mannose hydrolases are recovered trolled release pharmaceutical composition. The term “phar from the culture medium using known techniques and 40 maceutically acceptable' refers to molecules and composi treated with GlcNAc-phosphotransferase and phosphodi tions that are physiologically tolerable and do not typically ester C.-GlcNAcase according to the method herein to pro produce an allergic or Similar unwanted reaction Such as duce hydrolases that have M6P and can therefore bind to gastric upset or dizzineSS when administered. Preferably, membrane M6P receptors and be taken into the cell. “pharmaceutically acceptable” means approved by a regu Preferably, the cells are CHO cells and the hydrolases are 45 latory agency of the Federal or a State government or listed secreted with the MAN7(DD) structure. FIG. 3 shows the in the U.S. Pharmacopoeia or other generally recognized reaction Scheme for this method. pharmacopoeia for use in animals, preferably humans. The In a preferred embodiment, recombinant human acid term “carrier refers to a diluent, adjuvant, excipient, or alpha glucosidase (“rh-GAA") is prepared by culturing CHO vehicle with which the compound is administered. Such cells secreting rh-GAA in Iscove’s Media modified by the 50 pharmaceutical carriers can be Sterile liquids, Such as Saline addition of an alpha 1.2-mannosidase inhibitor. Immunopre Solutions, dextrose Solutions, glycerol Solutions, water and cipitation of rh-GAA from the media followed by digestion oils emulsions Such as those made with oils of petroleum, with either N-glycanase or endoglycosidase-H demonstrates animal, vegetable, or Synthetic origin (peanut oil, Soybean that in the presence of the alpha 1.2-mannosidase inhibitor oil, mineral oil, or Sesame oil). Water, Saline Solutions, the rh-GAA retains high mannose Structures rather than the 55 dextrose Solutions, and glycerol Solutions are preferably complex Structures found on a preparation Secreted in the employed as carriers, particularly for injectable Solutions. absence of the inhibitor. The secreted rh-GAA bearing high The enzyme or the composition can be administered by mannose Structures is then purified to homogeneity, prefer any Standard technique compatible with enzymes or their ably by chromatography beginning with ion exchange chro compositions. For example, the enzyme or composition can matography on ConA-Sepharose, Phenyl-Sepharose and 60 be administered parenterally, transdermally, or affinity chromatography on Sephadex G-100. The purified transmucosally, e.g. orally or nasally. Preferably, the enzyme rh-GAA is then treated in vitro with GlcNAc or composition is administered by intravenous injection. phosphotransferase to convert Specific mannoses to The following Examples provide an illustration of GlcNAc-phospho-mannose diesters. The GlcNAcphospho embodiments of the invention and should not be construed mannose diesters are then converted to M6P groups by 65 to limit the scope of the invention which is set forth in the treatment with phosphodiester a GlcNAcase. Experiments appended claims. In the following Examples, all methods show that 74% of the rh-GAA oligosaccharides were described are conventional unless otherwise Specified. US 6,534,300 B1 23 24 EXAMPLES Example 2 Materials and Methods Purification of Bovine GlcNAc-Phosphotransferase Lactating bovine udders were obtained from Mikkelson Lactating bovine mammary gland (6 kg) was collected at Beef, Inc. (Oklahoma City, Okla.). Ultrasphere ODS col Slaughter and immediately sliced into 10 cm thick Slices and umns were obtained from Beckman Instruments. Microsorb chilled in ice. Following homogenization in a Waring com MV-NH columns were obtained from Rainin Instrument mercial blender, the post-nuclear Supernatant fraction was Co., Inc. (Woburn, Mass.). Y PATP (7000 Ci/mmol; end prepared by centrifugation. Membrane fragments were col labeling grade), NaI, and Lubrol (CH3(CH2CH2O) lected by high speed centrifugation (39,000 xg, 45 minutes) H) were obtained from ICN (Costa Mesa, Calif.). Super and membrane proteins were solubilized in 4% Lubrol, 0.5% ose 6 (prep grade), DEAE-Sepharose FF, QAE-Sephadex deoxycholate. GlcNAc-phosphotransferase was specifically A-25, molecular mass standards for SDS-PAGE, HiTrap adsorbed from the solubilized membrane fraction by incu protein G columns, and Mono Q columns were obtained bation overnight with 10 ml of monoclonal antibody PT18 from Pharmacia Biotech Inc. 3M-Emphaze BioSupport coupled to UltralinkTM matrix (substitution 5 mg/ml). The Medium AB1, IODO GEN iodination reagent, and the BCA 15 matrix was then collected by low Speed centrifugation, protein assay reagent were obtained from Pierce. Glycerol, washed with 0.025 M Tris-HCI, pH 7.4, 0.005 M MgCl, Sucrose, C.-methylmannoside, C.-methylglucoside, reactive 0.3% Lubrol buffer containing 1 M NaCI. The column was green 19-agarose, Sodium deoxycholate, benzamidine, then washed with 2 column volumes of 0.01 M Tris-HCI, pH UDP-GlcNAc, phenylmethylsulfonyl fluoride, Tris, rabbit 7.4, 0.005 M MgC12, 0.3% Lubrol buffer. GlcNAc anti-mouse IgG, and mouse monoclonal antibody isotyping phosphotransferase was then eluted from the column with reagents were obtained from Sigma. 0.10 M Tris-HCl, pH 10.0, 0.005 M MgC12, 0.3% Lubrol POROS 50 HQ was obtained from PerSeptive Biosystems and neutralized with 1/10th volume of 1 M Tris-HCl, pH 6.0. (Cambridge, Mass.). ProBlott polyvinylidene difluoride Recovery is typically 20-50% of the GlcNAc membranes were obtained from Applied BioSystems Inc. phosphotransferase activity present in the homogenized (Foster City, Calif.). A Model QT12 rotary tumbler was 25 tissue, and approximately 0.5 mg of enzyme is recovered per obtained from LORTONE, Inc. (Seattle, Wash.). A mouse 10 kg of tissue processed. immunoglobulin Standard panel was obtained from Southern Biotechnology ASSociates, Inc. (Birmingham, Ala.). Recom Example 3 binant interleukin-6, porcine uteroferrin, and monoclonal antibody BP95 were gifts from colleagues. Other chemicals Amino Acid Sequencing of Bovine GlcNAc were reagent grade or better and were from Standard Sup phosphotransferase pliers. Example 3A Example 1 Preparation of Monoclonal Antibodies Specific for 35 Reduction, Alkylation and Separation of Individual Bovine GlcNAc-phosphotransferase Subunits Bovine GlcNAc-phosphotransferase was partially puri Bovine GlcNAc-phosphotransferase, 1.9 mg was desalted fied 30,000 fold as described (Bao, M., Booth J. L., et al. on a column of Sephadex G-25 Superfine equilibrated in 9% (1996). “Bovine UDP-N-acetylglucosamine: Lysosomal 40 formic acid and lyophilized. The lyophilized protein was enzyme N-acetylglucosamine-1-phosphotransferase. I. Puri dissolved in 1 ml of 500 mM Tris-HCl, pH 8.6, 6 M fication and subunit structure.” Journal of Biological Chem guanidine-HCl, 10 mM EDTA, 2 mM DTT degassed by istry 271: 31437-31445) and used to immunize mice. bubbling Ngas through the solution and incubated at 37 C. Spleens of immune mice were removed and Spenocytes for 1 hour. The Solution was made 5 mM in iodoacetic acid fused with SP2/0 myeloma cells according to Harlow 45 and incubated at 37 C. in the dark for a further 2% hours. (Harrow, E. and Lane, D. (1988). Antibodies: a laboratory The solution was then made 15 mM in B-mercaptoethanol manual, Cold Spring Harbor Laboratory). The fusion was and chromatographed on a column of SephadeX G-25 Super plated into 96 well plates and cultured in HAT media until fine equilibrated in 9% formic acid. The void fraction was hybridomas were visible. pooled and lyophilized. The individual subunits were Hybridomas Secreting monoclonal antibodies capable of 50 resolved by chromatography on a 1.0x30 cm column of capturing GlcNAc-phosphotransferase from a crude Sample Superose 12 equilibrated with 9% formic acid. were identified by incubation of hybridoma media (200 ul) with 200 units. Partially purified GlcNAc Example 3B phosphotransferase and capturing the resulting immune Amino Terminal Sequencing of Individual Subunits complex on rabbit anti-mouse IgG bound to protein A 55 coupled to UltralinkTM matrix. Immune complexes which Bovine GlcNAc-phosphotransferase, 0.5 mg was equili contained monoclonal antibodies directed against GlcNAc brated with Sodium dodecyl Sulfate, electrophoresed on a phosphotransferase were then identified by assay of the 6% polyacrylamide gel in the presence of Sodium dodecyl immune complex for GlcNAc-phosphotransferase activity. Sulfate. The resolved Subunits were then electro-transferred By this Strategy, four monoclonals directed against GlcNAc 60 to a PVDF membrane and the protein bands detected by phosphotransferase were identified in the fifth fusion Staining with Coomassie Blue. The bands corresponding to screened. The hybridomas identified were subcloned twice the individual Subunits were then excised with a razor blade using the same assay and ascites was produced in BALBc and Subjected to amino-terminal Sequencing in an Applied mice according to standard techniques (Harlow, E. and Lane, Biosystems Model 492 protein sequencer. The amino ter D. (1988). Antibodies: a laboratory manual, Cold Spring 65 minal Sequence of the C-Subunit was Met Leu Leu Lys Leu Harbor Laboratory). The monoclonal antibody labeled PT18 Leu Gln Arg Gln Arg Gln Thr Tyr (SEQ ID NO:26). The was Selected for use in further experiments. amino terminal sequence of the B Subunit is Asp Thr Phe Ala US 6,534,300 B1 25 26 Asp Ser Leu Arg Tyr Val ASn Lys Ile Leu ASn Ser Lys Phe The human B-subunit cDNA was cloned by screening a Gly Phe Thr Ser Arg Lys Val Pro Ala His (SEQ ID NO:27). size selected human placental cDNA library (Fischman, K., The amino terminal sequence of the Y-subunit is Ala Lys Met Edman J. C., et al. (1990). “A murinefer testis-specific Lys Val Val Glu Glu Pro ASn Thr Phe Gly Leu ASn ASn Pro transcript (ferTencodes a truncated fer protein.” Molecular Phe Leu Pro Gln (SEQ ID NO:28). and Cellular Biology 10: 146-153) obtained from ATCC with the random hexamer labeled murine f-subunit cl)NA Example 3C under conditions of reduced stringency (55° C., 2xSSC). Internal Amino Acid Sequence of the B- and Y The remaining portion of the C/B-subunit precursor cDNA Subunits was cloned by a combination of a walking Strategy begin 1O ning with the portion of the cDNA encoding the human The resolved ?- and Y-subunits from example 3B were B-Subunit and Standard library Screening Strategies. treated with trypsin at a 1/40 mass ratio overnight at 37 C. Additionally, EST data base searches were used to identify in 0.1 M Tris-HCl, pH 8.0. The tryptic fragments were then clones containing portions of the human C/B cDNA, which resolved by reverse phase chromatography on a C18 column were obtained from the corresponding repositories and equilibrated with 0.1% trifluoroacetic acid and developed 15 Sequenced. Together these Strategies allowed the determi with a linear gradient in acetonitrile. Well resolved peaks nation of the full length human C/B-Subunits precursor were then Subjected to amino terminal Sequencing as cDNA sequence. A clone containing this Sequence was described in example 3B. The peptides Sequenced from the assembled using the appropriate fragments and cloned into f3-subunit had the sequences Ile Leu ASn Ser Lys (SEQ ID pUC19. The 5597 bp sequence is given in Sequence NO:4 NO:29), Thr Ser Phe His Lys (SEQ ID NO:30), Phe Gly Phe and contains DNA sequences predicted to encode protein The Ser Arg (SEQID NO:31), and Ser Leu Val Thr Asn Cys Sequences homologous to all of the amino terminal and Lys Pro Val Thr Asp Lys (SEQ ID NO:32). The peptide internal peptide Sequences determined from the bovine C.- Sequenced from the Y-Subunit had the Sequence Leu Ala His and B-Subunits. Val Ser Glu Pro Ser Thr Cys Val Tyr (SEQ ID NO:33). A Second peptide Sequence from the Y-Subunit was obtained by Example 5 chymotryptic digestion with the Sequence ASn ASn Pro Phe 25 Leu Pro Gin Thr Ser Arg Leu Gin Pro (SEQ ID NO:34). Cloning the Human GlcNAc-phosphotransferase Y Subunit cDNA Example 3D The Y-Subunit amino terminal and tryptic peptide Internal Amino Acid Sequence of the C-Subunit Sequences were used to Search the Expressed Sequence Tag Internal peptide Sequences of the C-Subunit were obtained (EST) data base using the program thlastin. Altschul, S. F., as follows. Bovine GlcNAc phosphotransferase was Gish W., et al. (1990). “Basic Local Alignment Search reduced, alkylated, electrophoresed and transferred to PVDF Tool.” Journal of Molecular Biology 215: 403-10. Three as previously described. The C-Subunit band was excised human EST sequences were identified which were highly and tryptic peptides generated by in Situ digestion with 35 homologous to the determined bovine protein Sequences. trypsin, eluted with acetonitrile/trifluoroacetic acid and frac cDNA clone 48250 from which EST sequence 280314 was tionated by reverse phase HPLC. Individual peaks were then determined was obtained from Genome Systems and examined by Matrix ASSociated Laser Desorption Sequenced using Standard techniques. This clone contained Ionization-Mass Spectroscopy (MALDI-MS) and peaks a 1191 bp insert which contained all the determined protein containing a single mass were Subjected to amino terminal 40 Sequences and appeared to contain a Signal Sequence 5' of Sequencing as above. The peptide Sequences determined the determined amino terminal Sequence. The clone how from the C-Subunit are Val Pro Met Leu Val Leu Asp Xaa Ala ever lacked an initiator methionine or any 5' non-coding Xaa Pro Thr Xaa Val Xaa Leu Lys (SEQ ID NO:35) and Glu sequence. The 5' portion of the cDNA was obtained by PCR Leu Pro Ser Leu Tyr Pro Ser Phe Leu Ser Ala Ser Asp Val the C. W. C. S. C. primer Phe ASn Val Ala Lys Pro Lys (SEQ ID NO:36). 45 5'-GCGAAGATGAAGGTGGTGGAGGACC-3' (SEQ ID NO:37) and a T7 promoter primer were used in a reaction Example 4 along with template DNA from a human brain cDNA library in pCMV-SPORTOGIBCO). A 654 bp product was obtained, Cloning the Human GlcNAc-phosphotransferase C/ cloned in pCR2.1 and Sequenced. The Sequence demon |B-Subunit cDNA 50 strated the amplified product contained 23 bp of 5' non The amino-terminal protein Sequence determined from coding Sequence, the initiator methionine and the Signal the isolated bovine B-subunit was used to search the peptide identified in EST 280314. A full length cDNA for the Expressed Sequence Tag (EST) database using the program Y-subunit (pBC36) was assembled by ligating a 75 bp tblastin. Altschul, S. F., Gish W., et al. (1990). “Basic Local EcoRI-Apal fragment from the cloned PCR product, an Alignment Search Tool.” Journal of Molecular Biology 215: 55 ApaI-Not fragment from clone 48250 and EcoRI-Not cut 403-410. This search identified a partial mouse cDNA pcDNA3 (Invitrogen). previously identified during a positional cloning Strategy. Cordes, S. P. and Barsh, G. S. (1994). “The mouse segmen Example 6 tation gene kr encodes a novel basic domain-leucine Zipper Cloning the Human GlcNAc-phosphotransferase C/ transcription factor.” Cell 79: 1025-11034. 60 A forward PCR primer was designed based on the mouse B-Subunit Gene Sequence and used with an oligo dT reverse primer for Plasmid DNA was prepared from a human brain cDNA RT-PCR amplification of a 1,848 bp product using mouse library (Life Technologies) according to the manufacturers liver poly A RNA as template. The PCR product was cloned protocol. This DNA was used as template for PCR using and Sequenced and proved to contain all the determined 65 prime rS with the Sequence S B-Subunit Sequences, demonstrating it encoded the murine 5'-TGCAGAGACAGACCTATACCTGCC-3' (SEQ ID B-subunit. NO:38) and 5' ACTCACCTCTCCGAACTG-GAAAG-3' US 6,534,300 B1 27 28 (SEQ ID NO:39) using Taq DNA polymerase and buffer A MfeI-AgeI fragment derived from paD124). Plasmid from Fischer Scientific using 35 cycles of 94 C. 1 minute, pAD130 was then grown and Subsequent Sequencing of 55 C. 1 minute, and 79 C. 1 minute. A 106 bp product was plasmid pad 130 demonstrated that the AAAAA sequence obtained, purified by agarose gel electrophoresis, isolated by had reverted to AAAA again indicating instability in the GeneClean (Biol(01) and cloned into pCR2. DNA sequenc 5 Sequence at this point. ing determined the resulting plasmid pa)39 contained a In order to eliminate this instability the first AAA 106 bp insert which was excised by digestion with EcoRI (position 2761–2763 shown in SEQ ID NO:4) that codes for and Submitted to Genome Systems for Screening of a human lysine was changed to AAG (also coding for lysine) So that genomic BAC library. Four human BACs were identified the unstable AAAAA Sequence was changed to a stable and BAC #14951 was sequenced. For sequencing BAC AAGAA without altering the encoded amino acid. Plasmid #14951 was submitted to a colleague's laboratory at the pAD130 was corrected by removing a 214 bp Mfe-DraIII University of Oklahoma. The BAC was then fragmented by fragment and replacing it with a fragment with the correct nebulization, and fragments cloned into puC18 and Shotgun Sequence. The correct Mfe-Dral fragment was prepared Sequenced. Contigs were generated by computer analysis by PCR using paD130 as a template with forward primer and gaps closed by primer walking Strategies. The Sequence 15 5'-GAAGACACAATTGGCATACTTCACTGATAGCAA of the BAC spans 177,364 bp. The GlcNAc GAATACTGGGAGGC AACTAAAAGATAC-3' (SEQ ID phosphotransferase C/B-Subunits precursor gene spans ~80 NO:42) (oligo TTI 25 with desired AAGAA sequence as kb and is arranged as 21 exons. underlined) and C. W. C. S. C. primer 5'-ACTGCATATCCTCAGAATGG-3' (SEQ ID NO:43) Example 7 (oligo TTI 24). The PCR fragment was subcloned into the EcoRV site of pBluescript KS II(+) (Stratagene) generating Cloning the Human GlcNAc-phosphotransferase Y pMK16. The insert was sequenced for confirmation and the Subunit Gene 215 bp Mfe-DraIII fragment was prepared. To avoid Mfe The human Y-subunit gene was identified by blastin DraIII sites on the vector pcDNA 3.1 (+) (Invitrogen), the searching of the NCBI High Throughput Genomic Sequence 25 NheI-Xbal fragment was prepared from paD130 and Sub (HGTS) database with the full length human Subunit clDNA cloned into the Xbal site of puC19 (Life Technologies) to sequence. The search identified a clone HS316G12(gi construct pMK15. pMK15 was cleaved with Mfe and 4495.019) derived from human chromosome 16 which con DraIII and the 6317 bp fragment was purified and ligated tained the human Y-subunit gene. The human GlcNAc with the MfeI-DraIII fragment from pMK16 to form pMK19 phosphotransferase Y-Subunit gene spans about 12 kb and is containing the desired Stable Sequence in pUC19. arranged as 11 exons. Exons 1-3 and 4-11 are Separated by The corrected cDNA for the C/B subunit was excised from a large intron of about 9 kb. pMK19 as a Kpn-Xbal fragment and Subcloned between the KpnI and Xbal sites of pcDNA6/V5/His-A and desig Example 8 nated pMK25. Plasmid pMK25 containing the cDNA as 35 shown in SEQ ID NO:20 where the nucleotide sequence for Preparation of Modified Expression Plasmid for the the modified human C/B-subunit precursor cDNA is shown Human GlcNAc-phosphotransferase C/B-Subunits in nucleotides 1-3768. This Sequence corresponds to and is Precursor cDNA a modification of the nucleotide sequence 165-3932 shown in SEO ID NO:4. An expression vector for the GlcNAc-phosphotransferase 40 C/B cDNA was constructed in pcDNA3.1(+) as follows. Two upstream ATG's in the 5'-noncoding Sequence of the human Example 10 GlcNAc-phosphotransferase cDNA were removed and the Construction of Expression Vectors for Soluble, Kozak Sequence were modified as follows. Two fragments Human GlcNAc-phosphotransferase C/B Subunits from p AD 98, which was the human GlcNAc 45 phosphotransferase C/B cDNA cloned into pcDNA3.1 (+), Precursor cDNA were excised. A 1068 bp XhoI-Pst fragment and a 9746 bp Plasmid pMK19 was digested with BgIII (cutting at Nhe-XhoI fragment were ligated with oligonucleotides positions 255 and 2703 shown in SEQ ID NO:20) and with Se que n c e S self-ligated to reduce the length of the cDNA to be amplified 5'-CTAGCCACCATGGGGTTCAAGCTCTTGCA-3' (SEQ 50 from approx. 3.5 kb to 1 kb so that the 5' and 3' ends of the ID NO:40) and 5'-AGAGCTTGAACCCCATGGTGG-3' cDNA can be modified by PCR to remove the transmem (SEQ ID NO:41) generating paD105. The poly A sequence brane domains of the C. and B subunits of human GlcNAc near the 3' end of the cDNA clone was removed by ligating phosphotransferase and used to construct expression vectors a NheI-BgIII fragment from the cDNA with NheI-BamHI to produce Soluble GlccNAc-phosphotransferase. This plas cut vector pcDNA3.1 (+) generating paD 128. 55 mid was designated pMK21. The Strategy is that the nucle otides encoding the first 44 amino acids containing the Example 9 transmembrane domain of the a subunit (nucleotides 1-132 of SEQ ID NO:20) are replaced with a HindIII site, and Preparation of an Expression Plasmids for the nucleotides encoding the last 47 amino acids containing the Human GlcNAc-phosphotransferase C/B0 Subunits 60 transmembrane domain of the C. Subunit (nucleotides Precursor cDNA 3628-3768 of SEQ ID NO:21) are replaced with a stop DNA sequencing of paD128 identified deletion of an Ain codon and a Xbal site. an AAAAA sequence (positions 2761-2765 shown in SEQ Plasmid pMK21 was used as a template for PCR with the ID NO:4) that disrupted the coding sequence. Plasmid following primers: A forward primer (5'- pAD130 was constructed in an attempt to correct this by 65 TGGTTCTGAAGCTTAGCCGAGCCAGATCAATACC ligating a 5929 bp Nhe-Mfe fragment and a 2736 bp ATG-3' (SEQID NO:44), oligo TTI 76) containing a HindIII NheI-Age fragment (both from paD128 with a 515 bp Site (underlined) and a sequence complementary to nucle US 6,534,300 B1 29 30 otides 133 to 151 of SEQ ID NO:20 (italics), which will C GCC G G T G GTA C C CTA ATC GT CATC produce the 5'-end of a PCR fragment that removes the CGCGGAATAATCATACGCGTCA coding Sequence of the first 44 amino acids comprising the ACT CGGATTAT GT C TCGCA GAAGATCAGGTA putative transmembrane domain of the C. Subunit. A reverse GATCCGC GGTTAATCGACG primer (5'-TAGTACACTCTAGActactaCTTCAATTGTC TCGATAAG-3' (SEQ ID NO:45), oligo TTI 78) containing TGAGCC TAATA CAGAGC GT CTT CTA GT a Xbal site (underlined), two stop codons (lower case) and CCATCTAGGCGCCAATTAGCTGC a sequence complementary to nucleotides 3608 to 3627 of GTA SEQ ID NO:21 (italics), which will produce the 3'-end of a CATTCGA (SEQ ID NO:48) PCR fragment that removes the coding Sequence of the last The cDNA encoding “soluble C/B subunits' can be 47 amino acids comprising the putative transmembrane obtained as a HindIII-Xbal fragment from pMK49 and domain of the B Subunit and replaces it with two stop inserted into the plasmid pMK43 to ferm pMK50; pMK44 codons. The resulting PCR fragment was subcloned into the EcoRV site of pBluescript KS II+ (Stratagene). This to form pMK51, and into pMK45 to form pMK52, plasmids plasmid, designated pMK42, was Sequenced to ensure no capable of encoding the C/B subunits of human GlcNAc errors were introduced by PCR. The Bg|II-BgIII fragment 15 phosphotransferase with putative transmembrane domains (positions 255–2703 shown in SEQ ID NO:20) which was deleted, with different Signal peptides and all having the previously removed was subcloned back into the Bg|II site HPC4 epitope tag to facilitate purification of the soluble, of pMK42. The orientation of this fragment was determined Secreted enzyme. to be correct and this plasmid was designated pMK49. Thus, plasmid pMK49 contained a cDNA comprising a 5' HindIII Example 11 Site and a 3' Xbal Site flanking a coding region for the human Construction of Expression Vectors for the Human GlcNAc-phosphotransferase C/Bsubunits precursor cDNA GlcNAc-phosphotransferase y Subunit Precursor with the C. Subunit putative transmembrane domain deleted cDNA and the putative transmembrane domain of the B Subunit replaced with two stop codons (soluble C/B-cDNA). 25 The human GlcNAc-phosphotransferase Y-subunit pre This “soluble C/B-cDNA” can now be conveniently Sub cursor cDNA was obtained from plasmid paD133 in cloned into vectors constructed to contain the HPC4 epitope pAC5.1/V5-His by cutting with EcoRI. This cDNA was (used for rapid purification of the Soluble enzyme) and inserted into EcoRI digested pcDNA6/V5/His-A to form different secretion signal peptides. These pcDNA6/V5/His plasmid pMK17 containing cDNA as shown in SEQ ID A+tag) vectors were constructed as follows: NO:5. Plasmid pMKI7 was digested with Mlul (position Synthetic oligonucleotide cassettes containing a 5'-Nhe 124-129 as shown in SEQ ID NO:5) and EcoRI (position Site and a 3'-HindIII Site flanking nucleotide Sequences 1103-1108 as shown in SEQ ID NO:5) and the 980 bp coding for different secretion signal peptides and the nucle Mlu I-EcoRI fragment was then subcloned in otide Sequence coding for the HPC4 epitope were inserted pBluescriptKSII(+) with a synthetic double stranded cas into plasmid pcDNA6/V5/His-A cut with NheI and HindIII. 35 sette having an HindIII site and a Mlul site flanking a The following plasmids were prepared with the indicated nucleotide Sequence including positions corresponding to CaSSette: 95-123 as shown in SEQ ID NO:5 thereby removing the 1. pMK45- mouse immunoglobulin Kappa chain Signal nucleotide Sequence encoding the amino terminal, 24-amino peptide (sequence in italics) and HPC4 epitope (Sequence acid signal peptide in plasmid pMK26. Plasmid pMK26 was underlined) 40 Sequenced to ensure its Sequence. The correct cDNA from CTAGCCGCCACC ATGGA GACAGACACACTC pMK26, which encodes amino acids for the human GlcNAc CTGCTA TGGGTA CTGCTGCTC phosphotransferase y Subunit with the Signal peptide GGCGGTGGTACC TC TGTCT GTGTGAGGAC removed, is then excised from pMK26 by HindIII and EcoRI GATACCCATGACGACGAG digestion and placed into plasminds pMK43 to form 45 pMK58; pMK44 to form pMK59, and into pMK45 to form TGGGTTCC AGGT TC CACTGGTGA CGAAGAT pMK64, plasmids capable of encoding the Y subunit of CAGGTAGATCCGCGGTTAATC human GlcNAc-phosphotransferase with its signal peptide ACCCAAG GTCCAAG GTGACCACTG CTTC deleted, with different Signal peptides and all having the TAGTCCAT CTAGGCGCCAATTAG HPC4 epitope tag to facilitate purification of the soluble, Y GACGGTA Subunit. CT GCCATTCGA (SEQ ID NO:46) 50 1. pMK44-a transferrin signal peptide sequence (in italics) To evaluate the behavior of C/B/y secreted products, the and HPC4 epitope (sequence underlined) C/B Subunit precursor and the Y Subunit were co-expressed CTAGCGGTACCATGAGATT AGCAGTAGGCGCC in the bi-cistronic vector pIRES (Clontech). This was TTATTAG TATGCGC AGTACT C accomplished by Subcloning C/B and Y cDNAS expressing 55 the desired Subunit with a Selected Signal peptide and the CGCC ATG GTACT CTAATCGTCATCC GCG HPC4 Tag into NheI site (MCS-A) and Xbal site (MCS-B) GAATAATCATACGCGTCATGAG of plRES, respectively. GGATTAT GTC TCGCAG AAGATCAGGTAGATC CGC GGTTAATCGACGGTA Example 12 CCTTATACAGAGCGTCTTCTAG TCCAT CTAG 60 GCGCCAATTAGCTGCCATTCGA Transient Expression of the C/B and Y Subunits of (SEQ ID NO:47) Human GlcNAc-phosphotransferase in 293T Cells 1. pMK43-a transferrin Secretion peptide Sequence modi Plasmids were transfected into 293T cells using Fugene6 fied to satisfy a Kozak's sequence(sequence in italics) and (Roche) according to manufacturer's instructions. Culture HPC4 epitope (Sequence underlined), 65 media was collected 23 h, 44.5 h and 70 h after transfection. CTAGCCGCCACCATGGGATT AGCAGTAGGCGC Aliquots of media containing expressed protein was cap CTTATTAGTATGCGC AGT tured on anti-HPC4 monoclonal antibody (U.S. Pat. No. US 6,534,300 B1 31 32 5,202,253) conjugated with Ultralink beads (Pierce) by of mice with a partially purified preparation of phosphodi overnight incubation at 4 C. The beads were washed to estcr C.-GlcNAcase. Spleens were then removed from remove unbound protein and assayed directly for phospho immune mice and fused with SP2/O myeloma cells accord transferase activity as described previously (REF). ing to standard techniques (Harrow, E. and Lane, D. (1988). Plasmids used for expression all containing a Sequence 5 Antibodies: a laboratory manual, Cold Spring Harbor encoding for the HPC4 tag were as follows: Laboratory). Hybridomas were plated in eight 96-well plates 1. pMK50-modified transferrin secretion peptide and and grown until hybridomas were visible. Hybridomas C/B/y subunit in pcDNA6/V5/His-4 Secreting antibodies to phosphodiester C.-GlcNAcase were 2. pMK51-transferrin secretion peptide and C/B subunit identified measuring phosphodiester O-GlcNAcase activity in pcDNA6/V5/His-4 in immunoprecipitates prepared by incubation of a partially 3. pMK52-mouse immunoglobulin secretion peptide purified preparation of phosphodiester C.-GlcNAcase with and C/B subunit in pcDNA6/V5/His-4 pooled hybridoma Supernatants. Pools from 16 and 4 wells 4. pMK75-modified transferrin secretion peptide and were assayed followed by individual wells. Monoclonal C/B Subunit and modified transferrin Secretion peptide UC1 was identified by this protocol and coupled to and Y subunit in pRES 15 UltralinkTM for use in purification of phosphodiester 5. pMK81-transferrin secretion peptide and C/B subunit O-GlcNAcase. and transferrinsecretion peptide and Y subunit in pRES 6. pMK76-mouse immunoglobulin secretion peptide Example 15 and C/B Subunit and mouse immunoglobulin Secretion peptide and Y in pRES Purification of Bovine Phosphodiester C.- The relative amounts of expression detected by assay for GlcNAcase phosphotransferase using methyl-O-D-mannoSide and UDP Bovine calf liver (1 kg) was homogenized in 0.05 M B-P-GlcNAc as substrates with cell transfected with Imidazole-HCI, pH 7.0, 0.15 M NaCI, 0.01 M EDTA and a pcDNA6/V5/His-4 as controls is shown in FIG. 4. washed post-nuclear Supernatant was prepared. Membranes Example 13 25 were collected by centafugation at 30,000xg for 30 minutes and washed three times with the above buffer. Membrane Expression and Purification GlcNAc proteins were then solubilized in buffer containing 2% phosphotransferase C/B/Y Triton X-100, 0.05% deoxycholate and insoluble material For expression and purification of the enzyme, a modified removed by centrifugation, as before. The solubilized mem expression plasmid is constructed in a modified expression brane fraction was incubated with 20 ml of monoclonal vector derived from p. 14. The plasmid directs the synthe antibody UC1 coupled to UltralinkTM (substitution 5 mg/ml) sis of a Soluble epitope tagged GlcNAc-phosphotransferase with constant rotation for 16 hours at 4 C. The UC1 molecule. The C/B-subunit precursor is modified as follows: UltralinkTM was collected by low speed centrifugation. The 5' portion of the cDNA which encodes the C-Subunit packed into a column and washed with 0.025 M Tris-HCI, cytoplasmic and transmembrane domain is deleted and 35 pH 7.4, 0.3% Lubrol, followed by two column volumes of replaced with nucleotides which encode the transferrin Sig 0.5 M NaHCO3, pH 8.0, 0.3% Lubrol. Phosphodiester nal peptide followed by amino acids which encode the O-GlcNAcase was then eluted with 0.5 M NaHCO3, pH epitope for monoclonal antibody HPC4. The 3' portion of the 10.0, 0.3% Lubrol and collected in 1/10 volume of 1.0 M cDNA is modified by the insertion of a stop codon before the Tris-HCI, pH 5.5. B-Subunit transmembrane Segment. The vector peE14.1 40 (Lonza Biologics) is modified by the insertion of a 850 bp Example 16 Mlu-Nicol fragment containing a modified vascular endot helial growth factor (VEGF) promoter at the unique Mlul Amino Acid Sequencing of Bovine Phosphodiester site in pEE 14.1. This vector encoding the modified GlcNAc O-GlcNAcase phosphotransferase C/B-Subunit precursor is co-transfected 45 Example 16A with a wild type Y-subunit construct containing the VEGF promoter in peE14.1 into CHO-K1 cells using Fugene6 and Amino-terminal Sequence of Bovine plated into 96 well plates. Transfectants are selected in 25 Phosphodiester C.-GlcNAcase tum methionine Sulfoximine and the plasmid amplified by Bovine phosphodiester O-GlcNAcase was bound to a 0.25 selection in 96 well plates with 50 uM, 100 uM,250 uM, and 50 ml column of POROS HQ and step-eluted with buffer 500 uM methionine Sulfoximine. Clones are picked into containing 0.5 M NaCl. Fractions containing phosphodiester duplicate 96 well plate and the highest expressing clones O-GlcNAcase activity were identified by phosphodiester Selected by dot blotting media and immuno-detection with O-GlcNAcase assay, pooled and absorbed to a ProSorb monoclonal antibody HPC4. The highest expressing clone is Sample Preparation Cartridge (Perkin Elmer) and subjected expanded into cell factories. The recombinant soluble 55 to amino acid Sequencing in an Applied BioSystems Model epitope tagged GlcNAc-phosphotransferase is purified from 492 Protein Sequencer operated according to the manufac the media by chromatography on monoclonal antibody turer's instructions. The Sequence Asp-Xaa-Thr-Arg-Val HPC4 coupled to Ultralink in the presence of 5 mM MgCl His-Ala-Gly-Arg-Leu-Glu-His-Glu-Ser-Trp-Pro-Pro-Ala and 1 mM CaCl. The soluble epitope tagged GlcNAc Ala-Gln-Thr-Ala-Gly-Ala-His-Arg-Pro-Ser-Val-Arg-Thr phosphotransferase is eluted with 5 mM EGTA and 5 mM MgCl. 60 Phe-Val was obtained. Example 14 Example 16B Preparation of Monoclonal Antibodies Specific for Internal Sequence of Bovine Phosphodiester C.- Bovine Phosphodiester C.-GlcNAcase 65 GlcNAcase Murine monoclonal antibodies specific for bovine phos Bovine liver phosphodiester C.-GlcNAcase was concen phodiester O-GlcNAcase were generated by immunization trated to 10 ul in a Speed Vac, combined with 30 ul 0.1 M US 6,534,300 B1 33 34 Tris-HCI, pH 7.4, 8 M guanidine-HCI, and 2–4 ul 25 mM they reached 50-80% confluence. The plates were then DTT and incubated at 50 C. for 1 hour. lodoacetamide 2.4 washed with OptiMEM I and the cells transfected with the lul 50 uM was then added and the incubation was continued expression vector described in Example 19 using Lipo for 1 hour. The reaction mixture was then desalted on a fectamine Plus (GIBCO BRL Life Technologies) according column of Sephadex G25 Superfine as described for to the manufacturers instructions. Cells were harvested at 48 GlcNAc-phosphotransferase and digested with trypsin. The hours, a Solubilized membrane fraction prepared and peptides were fractionated by HPLC and Sequenced as assayed for phosphodiester C-GlcNAcase activity. described for GlcNAc-phosphotransferase. The Sequences determined are Arg Asp Gly Thr Leu Val Thr Gly Tyr Leu Example 21 Ser Glu Glu Glu Val Leu Asp Thr Glu ASn and Gly Ile ASn 1O Leu Trp Glu Met Ala Glu Phe Leu Leu Lys. Expression and Purification of Soluble Example 17 Recombinant Human phosphodiester C.-GlcNAcase For expression and purification of the enzyme, a modified Cloning the Human Phosphodiester O-GlcNAcase expression plasmid is constructed in a modified expression cDNA 15 vector derived from peE14.1. The plasmid directs the syn The phosphodiester C.-GlcNAcase tryptic peptide thesis of a Soluble epitope tagged phosphodiester C.GlcNA Sequences were used to Search the EST data bases as case molecule. The phosphodiester O-GlcNAcase precursor described for GlcNAc-phosphotransferase above. Three is modified as follows: The 3' portion of the cDNA which EST sequences were identified which contained the human encodes the phosphodiester C.-GlcNAcase transmembrane phosphodiester C.-GlcNAcase cDNA and clone ATCC and cytoplasmic domains is deleted and replaced with #367524 was obtained and a -700 bp EcoRI-NotI fragment nucleotides which encode the epitope for monoclonal anti was excised from this clone and used to probe a human liver body HPC4 followed by a stop codon. The vector p E14.1 cDNA library in the vector Trip1EX. Several clones were (Lonza Biologics) is modified by the insertion of a 850 bp identified and Sequenced, one of which (clone 6.5) proved to Mlu-Nicol fragment containing a modified vascular endot contain a nearly full length cDNA for the human phosphodi 25 helial growth factor (VEGF) promoter at the unique Mlul ester C.-GlcNAcase. The genomic clone described in Site in peE14.1. This vector encoding the epitope tagged Example 18 demonstrated that clone 6.5 was missing only Soluble phosphodiester C-GlcNAcase precursor is trans the initiator methionine. fected into CHO-K1 cells using Fugene6 and plated into 96 Example 18 well plates. Transfectants are Selected in 25 um methionine sulfoximine, and the plasmid amplified by selection in 96 Cloning the Human Phosphodiester O-GlcNAcase well plates with 50 uM, 100 uM, 250 uM, and 500 uM Gene methionine Sulfoximine. Clones are picked into duplicate 96 The human phosphodiester C.-GlcNAcase gene was iden well plate and the highest expressing clones Selected by dot tified by searching the NCBI database nr with the human blotting media and immuno-detection with monoclonal anti phosphodiester O-GlcNAcase cDNA using the program 35 body HPC4. Media from clones demonstrating the highest blastin. The genomic Sequence was determined during the level of epitope tag expression is assayed for phosphodiester Sequencing of a clone from chromosome 16p13.3 and O-GlcNAcase activity. The highest expressing clone is deposited Mar. 06, 1999 in GenBank as an unidentified expanded into cell factories. The recombinant soluble Sequence of 161264 bp with the accession number epitope tagged phosphodiester C-GlcNAcase is purified AC007011. The gene spans about 12 kb of genomic DNA on 40 from the media by chromatography on monoclonal antibody chromosome 16.13 and is arranged in 11 exons. HPC4 coupled to UltralinkTM in the presence of 5 mM MgCl2 and 1 mM CaCl2. The Soluble epitope tagged phos Example 19 phodiester C.-GlcNAcase is eluted with 5 mM EGTA and 5 Construction of an Expression Vector for Human mM MgCl2. Phosphodiester or O-GlcNAcase 45 An expression vector for human phosphodiester Example 22 O-GlcNAcase was prepared as follows: The 5' end of the Construction of an Expression Vector for Soluble, sequence of clone 6.5 was modified by PCR amplification of Human Phosphodiester O-GlcNAcase the 5' end of the cDNA with a forward primer with the 50 sequence 5'-GGAATTCCACCATGGCGACCTCCACGG For expression and purification of the enzyme, a modified GTCG-3' (SEQ ID NO:49) and a reverse primer expression plasmid is constructed in a modified expression 5'-TGACCAGGGTCCCGTCGCG-3' (SEQ ID NO:49). vector derived from the pEE14.1 vector (Lonza Biologics). This Served to add a consensus Kozak Sequence and initiator The plasmid directs the Synthesis of a Soluble epitope tagged methionine to the sequence of clone 6.5. The -500 bp PCR 55 phosphodiester O-GlcNAcase molecule. The phosphodiester product was purified, digested with EcoRI and BamHill and O-GlcNAcase precursor is modified as follows: The 3' ligated into pcDNA3.1 (-) which was sequenced. This portion of the cDNA (1342–1548 of SEQ ID NO:7) which construct was then digested with BamHI and HindIII and encodes the phosphodiester C.-GlcNAcase transmembrane ligated with a ~1600 bp BamHI-HindIII fragment containing and cytoplasmic domains was deleted and replaced with the 3' portion of the cDNA from clone 6.5 generating the full 60 nucleotide sequence GAGGACCAGGTGGACCCCAG length expression plasmid. GCTGATCCAC GGCAAGGAT (SEQ ID NO:51) that Example 20 encodes the epitope for monoclonal antibody HPC4 (EDQVDPRLIDGKD (SEQ ID NO:52)) followed by a stop Host Cell Preparation for Human Phosphodiester codon. O-GlcNAcase 65 This expression vector was constructed by generating two Cos cells were grown in 60 mm plates in Dulbeccos intermediate plasmids and ligating a fragment from each minimal essential media (DMEM) at 37°C. in 5% CO until into pEE14.1 vector (Lonza Biologics) to yield the final US 6,534,300 B1 35 36 expression vector. The first intermediate plasmid designated Example 24 pKB4 was constructed by ligating the 1034 bp FseI-Bsu36I fragment of phosphodiester O-GlcNAcase Growth of CHO Cells Expressing Recombinant (lacking the C-terminal transmembrane and cytoplasmic Human Acid C-glucosidase in the Presence of C-1, domains) from clone 6.5, and a Bsu36I-Xbal oligonucle 2 Mannosidase Inhibitors otide fragment that contains the HPC4 epitope into a modi fied puC19 vector. The second intermediate plasmid desig CHO cells expressing human acid C-glucosidase were nated pKB5, was constructed by ligating a 850 bp Mlul cultured in Glasgow Modified Minimal Essential Media Nicol fragment containing a modified vascular endothelial containing 5% Fetal Bovine Serum, 25 uM methionine growth factor (VEGF) promoter from pcDNA4/HisMax sulfoximine, 20 mM TES, pH 7.2, and 7.5 mM (Invitrogen), a 256 bp Bsel-Fsel fragment encoding the 1-deoxymannojirimycin-HC1. Alternatively, the cells can be N-terminus of human phosphodiester C.-GlcNAcase from cultured in the above media containing 100 tug/mL clone 6.5, and an oligonucleotide linker into a modified 1-deoxymannojirimycin-HC1 and 25 ug/mL kifunensine. pUC19 vector. The final expression vector designated pKB6 was constructed by ligating the Mlul-Fse fragment from 15 Example 25 pKB5, and the FSeI-HindIII fragment from pKB4 into a Mlul/HindIII digested peE14.1 vector. The plasmid pKB6 Isolation of Recombinant Human Acid C.- contains the nucleotide sequence shown in SEQ ID NO:22. glucosidase Expression and Purification of Soluble Recombinant Human Phosphodiester O-GlcNAcase Recombinant human acid C-glucosidase was purified from spent tissue culture media as follows: Media was Approximately 10, 293T cells were plated in a cell concentrated 10 fold by tangential ultrafiltration with a factory using Dulbecco's modified eagle's medium 30,000 dalton cutoff membrane and dialyzed into 50 mM (DMEM) containing 10% fetal bovine serum in a humidified Sodium phosphate, pH 6.5, and applied to a column of ConA atmosphere at 37° C. with 5% CO2. These cells were 25 Sepharose (Pharmacia). Following a wash with the same transfected with approximately 700 g of pKB6 using 2 ml of buffer to remove the unbound proteins, acid C-glucosidase transfection reagent Fugene-6 (Roche) for the transient was eluted with 1.0 M C.-methyl glucoside, pooled, concen expression of Soluble human phosphodiester C.-GlcNAcase. trated and dialyzed as before. The acid O-glucosidase was After three days of culturing the transfected cells, the then applied to a column of Sephadex G-200 equilibrated medium containing Soluble, epitope-tagged, human phos with 50 mM sodium phosphate, pH 6.5 and eluted isocrati phodiester O-GlcNAcase was collected and applied in the cally with the same buffer. presence of 1 mM CaCl2 to a column of monoclonal antibody HPC4 coupled to Ultralink (Pierce). Affinity purified, epitope-tagged, human phosphodie Ster Example 26 O-GlcNAcase (approximately 11 mg) was eluted with buffer 35 containing 5 mM EDTA and stored at -20° C. in 50 mM Treatment of Recombinant Human Acid C.- Tris, 150 mM NaCl, 2 mM CaC2, 50% glycerol, pH 7.2. The glucosidase with GlcNAc-phosphotransferase and enzyme had a specific activity of 500,000 units/mg with Phosphodiester C.-GlcNAcase HGlcNAc-phosphomannose-O-methyl as a substrate 40 Human acid C-glucosidase at 10 mg/ml was incubated in (Komfeld R, et al., JBC 273:23203–23210). 50 mm Tris-HCI, pH 6.7, 5 mM MgCl, 5 mM MnC1, 2 mM Example 23 UDP-GlcNAc with GlcNAc-phosphotransferase at 100,000 u/mL at 37 C. for 2 hours. Phosphodiester C.-GlcNAcase, CHO Cells Expressing Recombinant Human Acid 1000 u/mL was then added and the incubation continued for C-glucosidase 45 another 2 hours. The acid C-glucosidase was then repurified The human acid O-glucosidase cDNA was obtained from by chromatography on Q-Sepharose, and Step elution with Dr. Frank Martinuk (Martiniuk, F., Mehler, M., Tzall, S., NaCl. Meredith, G. and Hirschhorn, R. (1990). “Sequence of the cDNA and 5'-flanking region for human acid alpha Example 27 glucosidase, detection of an intron in the 5' untranslated 50 leader Sequence, definition of 18-bp polymorphisms, and Characterization of the Oligosaccharide Structures differences with previous cDNA and amino acid Sequences.” on Modified Recombinant Human Acid C.- DNA Cell Biol 9(2): 85–94) and cloned into the expression glucosidase vector p. 14.1. This vector was used to transfect CHO-K1 cells using Fugene6 and plated into 96 well plates. Trans 55 Recombinant acid O-glucosidase treated or untreated with fectants were Selected in 25 um methionine Sulfoximine, and GlcNAc-phosphotransferase and phosphodie Ster clones picked and plated into 96 well plates. The plasmid O-GlcNAcase was digested with N-glycanase (New England was amplified by selection with 50 uM, 100 uM, 250 uM, Biolabs) or endomannosidase H (New England Biolabs) and 500 uM methionine Sulfoximine. Clones were picked according to the manufacturer's conditions. The released into duplicate 96 well plates and the highest expressing 60 oligosaccharides were then labeled on the reducing terminus clones Selected by assay of the media for acid C-glucosidase with 2-aminobenzamide and fractionated by HPLC with activity and the cells for DNA content. The highest express fluorescent detection according to the manufacturer's ing clone (Clone 3.49.13) based on acid C-glucosidase instructions (Oxford Glycosystems). Peaks were identified activity to DNA content ratio was then expanded into a cell by comparison with Standards chromatographed on the same factory. This clone was incubated at 37 C. in 5% CO and 65 System, and confirmed by digestion with linkage specific maintained in Glasgow Minimal ESSential Media containing glycosidases and/or mass determination by MALDI. The 20 mM TES, pH 7.2, 5% fetal bovine serum. results are shown in Table 1. US 6,534,300 B1 38

TABLE 1. Enzyme Preparation M6 M7 M8 M9 IP-Gn. 2P-Gn IM6P Complex Rh-GAA O O O O O O 1. 99 (Secreted) Rh-GAA 23 31 23 6 O O 17 O (dMM/intracellular) Rh-GAA 6 11 7 2 12 62 O O (dMM/intracellular) Ptase-treated

Referring to Table 1, the data (given in mole percent) in 6 well plates and incubated at 37° C. in 5% CO. show that the LySOSomal enzymes prepared using the 15 Recombinant human acid O-glucosidase with different car GlcNAc-phosphotransferase and phosphodie Ster bohydrate Structures are compared for the rate and extent of O-GlcNAcase of the present invention are highly phospho rylated The data shows that the present invention produces internalization. Controls include each preparation incubated lysosomal enzymes having about 5-10 M6P groups per with 5 mM mannose 6-phosphate and incubations without enzyme compared to about 0–2 for untreated enzymes and added recombinant human acid C-glucosidase. The different enzymes known in the art. When compared to naturally preparations to be examined include acid C-glucosidase occurring or recombinant lySOSomal enzymes, the in vitro Secreted from CHO cells, acid C-glucosidase Secreted from modified preparation is very highly phosphorylated. In the CHO cells in the presence of C.1.2-mannosidase inhibitors, most highly phosphorylatedly SOSomal enzyme known in the acid C-glucosidase Secreted from CHO cells in the presence art, the O-galactosidase A described by Matsuura, F., Ohta, of C.1.2-mannosidase inhibitors treated with GlcNAc M., Ioannou, Y. A. and Desnick. R. J. (1998). “Human 25 phosphotransferase, and acid C-glucosidase Secreted from alpha-galactosidase A: characterization of the N-linked oli CHO cells in the presence of or 1.2-mannosidase inhibitors gosaccharides on the intracellular and Secreted glycoforms treated with GlcNAc-phosphotransferase and phosphodi overexpressed by Chinese hamster ovary cells.” Glycobiol ogy 8(4): 329-39, 5.2% of the oligosaccharides are bis ester C.-GlcNAcase. Equal amounts of the four different phosphorylated. In marked contrast, 62% of the oligosac preparations are added to each well and incubated at 37 C. charides on the in Vitro-phosphorylated acid C-glucosidase, for periods varying from 5 minutes to 4 hours. At the end of preparation described here contains bis-phosphorylated oli each incubation period the cell monolayers are washed with gosaccharides. This represents about a 12 fold increase. phosphate buffered Saline containing 5 mM mannose When the in vitro phosphorylated preparation of rh-GAA 6-phosphate and the monolayer solubilized in 1% Triton shown in Table 1 is compared with GAA secreted from CHO 35 X-100 and assayed for internalized acid C-glucosidase by cells by methods known in the art, an even greater increase enzymatic assay. in phosphorylation is evident, about a 62 fold increase. Thus, the in vitro-phosphorylated GAA is 12-62 fold more phosphorylated than any other described preparation of natural or recombinantlySOSomal enzyme. This difference 40 Applicant and the assignee acknowledge their responsi has a major influence on the rate and extent of internalization bility to replace these cultures should they die before the end (Reuser, A.J., Kroos, M.A., Ponne, N.J., Wolterman, R. A., of the term of a patent issued hereon, 5 years after the last Loonen, M. C., Busch, H. F., Visser, W.J. and Bolhuis, P. A. request for a culture, or 30 years, whichever is the longer, (1984). “Uptake and stability of human and bovine acid and their responsibility to notify the depository of the alpha-glucosidase in cultured fibroblasts and Skeletal muscle issuance of Such a patent, at which time the deposit will be cells from glycogenosis type II patients.” Experimental Cell made irrevocably available to the public. Until that time the Research 155:178–189). deposit will be made available to the Commissioner of Example 28 Patents under the terms of 37 C.F.R. 1.14 and 35 U.S.C. 112. Comparison of Cell Uptake of Recombinant 50 Human Acid C-glucosidase with or Without Modification by GlcNAc-phosphotransferase and While the preferred embodiments are shown to illustrate the invention, numerous changes to the materials and meth Phosphodiester C.-GlcNAcase ods can be made by those skilled in the art. All Such changes Human Pompe disease fibroblasts are obtained from are encompassed within the Spirit of the invention as defined ATCC and cultured in DMEM with 10% fetal bovine Serum by the appended claims.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 52

<21 Oc SEQ ID NO 1 <211 LENGTH 928 TYPE PRT US 6,534,300 B1 39 40

-continued <213> ORGANISM: Homo sapiens <400 SEQUENCE: 1 Met Leu Phe Lys Leu Leu Glin Arg Glin Thr Tyr Thr Cys Leu Ser His 1 5 10 15 Arg Tyr Gly Leu Tyr Val Cys Phe Leu Gly Val Val Val Thr Ile Val 2O 25 30 Ser Ala Phe Glin Phe Gly Glu Val Val Leu Glu Trp Ser Arg Asp Gln 35 40 45 Tyr His Val Lieu Phe Asp Ser Tyr Arg Asp Asn. Ile Ala Gly Lys Ser 50 55 60 Phe Glin Asn Arg Leu Cys Leu Pro Met Pro Ile Asp Val Val Tyr Thr 65 70 75 8O Trp Val Asn Gly Thr Asp Leu Glu Lieu Lleu Lys Glu Lieu Glin Glin Val 85 90 95 Arg Glu Gln Met Glu Glu Glu Gln Lys Ala Met Arg Glu Ile Leu Gly 100 105 110 Lys Asn. Thir Thr Glu Pro Thr Lys Lys Ser Glu Lys Glin Leu Glu Cys 115 120 125 Leu Lieu. Thir His Cys Ile Lys Val Pro Met Leu Val Lieu. Asp Pro Ala 130 135 1 4 0 Leu Pro Ala Asn Ile Thr Leu Lys Asp Val Pro Ser Leu Tyr Pro Ser 145 15 O 155 160 Phe His Ser Ala Ser Asp Ile Phe Asn. Wall Ala Lys Pro Lys Asn Pro 1.65 170 175 Ser Thr Asn Val Ser Val Val Val Phe Asp Ser Thr Lys Asp Val Glu 18O 185 190 Asp Ala His Ser Gly Lieu Lleu Lys Gly Asn. Ser Arg Glin Thr Val Trp 195 200 2O5 Arg Gly Tyr Leu Thir Thr Asp Lys Glu Val Pro Gly Leu Val Leu Met 210 215 220 Gln Asp Leu Ala Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys Glu Thr 225 230 235 240 Asn Gln Leu Lys Thr Lys Lieu Pro Glu Asn Lieu Ser Ser Lys Wall Lys 245 250 255 Leu Lieu Gln Leu Tyr Ser Glu Ala Ser Val Ala Leu Lleu Lys Lieu. Asn 260 265 27 O Asn Pro Lys Asp Phe Glin Glu Lieu. Asn Lys Glin Thr Lys Lys Asn Met 275 280 285 Thir Ile Asp Gly Lys Glu Lieu. Thir Ile Ser Pro Ala Tyr Lieu Lleu Trp 29 O 295 3OO Asp Leu Ser Ala Ile Ser Glin Ser Lys Glin Asp Glu Asp Ile Ser Ala 305 310 315 320 Ser Arg Phe Glu Asp Asn. Glu Glu Lieu Arg Tyr Ser Lieu Arg Ser Ile 325 330 335 Glu Arg His Ala Pro Trp Val Arg Asn Ile Phe Ile Val Thr Asin Gly 340 345 350 Glin Ile Pro Ser Trp Leu Asn Leu Asp Asin Pro Arg Val Thr Ile Val 355 360 365 Thr His Glin Asp Val Phe Arg Asn Leu Ser His Leu Pro Thr Phe Ser 370 375 38O Ser Pro Ala Ile Glu Ser His Ile His Arg Ile Glu Gly Lieu Ser Glin 385 390 395 400 US 6,534,300 B1 41 42

-continued

Phe Ile Teu Asn Asp Asp Wall Met Phe Gly Lys Asp Wal Trp 405 410 415

Pro Asp Asp Phe Ser His Ser Lys Gly Glin Lys Wall Tyr Telu Thr 420 425 430

Trp Pro Wall Pro Asn Ala Glu Gly Cys Pro Gly Ser Trp Ile Lys 435 4 40 4 45

Asp Gly Tyr Asp Ala Asn Asn Ser Ala Asp Trp Asp 450 455 460

Gly Gly Asp Ser Gly Asn Ser Gly Gly Ser Arg Ile Ala Gly 465 470 475 480

Gly Gly Gly Thr Gly Ser Ile Gly Wall Gly His Pro Trp Glin Phe Gly 485 490 495

Gly Gly Ile Asn Ser Wall Ser Cys Asn Glin Gly Cys Ala Asn Ser 5 OO 505 510

Trp Telu Ala Asp Phe Asp Glin Ala Asn Wall Teu Ser Cys 515 525

Gly Phe Asp Ala Gly Asp Cys Gly Glin Asp His Phe His Glu Telu Tyr 530 535 540

Lys Wall Ile Telu Teu Pro Asn Glin Thr His Tyr Ile Ile Pro Lys Gly 545 550 555 560

Glu Telu Pro Tyr Phe Ser Phe Ala Glu Wall Ala Arg Gly Wall 565 570 575

Glu Ala Tyr Ser Asp Asn Pro Ile Ile Arg His Ala Ser Ile Ala 585 59 O

Asn Trp Thr Ile His Telu Ile Met His Ser Gly Met Asn Ala 595 600 605

Thr Thr Ile His Phe Asn Teu Thr Phe Glin Asn Thr Asn Asp Glu Glu 610 615

Phe Met Glin Ile Thr Wall Glu Wall Asp Thr Arg Glu Pro Lys 625 630 635 640

Teu Asn Ser Thr Ala Glin Gly Tyr Glu Asn Teu Wall Ser Pro Ile 645 650 655

Thr Telu Telu Pro Glu Ala Glu Ile Telu Phe Glu Asp Ile Pro Lys Glu 660 665 670

Arg Phe Pro Phe Arg His Asp Wall Asn Ser Thr 675 680 685

Ala Glin Glu Glu Wall Ile Pro Telu Wall Asn Ile Ser Teu Telu Pro 69 O. 695 7 OO

Lys Asp Ala Glin Teu Ser Teu Asn Thr Telu Asp Teu Glin Teu Glu His 705 710 715 720

Gly Asp Ile Thr Teu Gly Tyr Asn Telu Ser Ser Ala Telu Telu 725 730 735

Arg Ser Phe Telu Met Asn Ser Glin His Ala Ile Asn Glin Ala 740 745 750

Ile Ile Thr Asp Glu Thr Asn Asp Ser Telu Wall Ala Pro Glin Glu Lys 755 760 765

Glin Wall His Ser Ile Teu Pro Asn Ser Teu Gly Wall Ser Glu Arg 770 775 78O

Teu Glin Arg Telu Thr Phe Pro Ala Wall Ser Wall Wall Asn Gly His 785 790 795

Asp Glin Gly Glin Asn Pro Pro Telu Asp Telu Glu Thr Thr Ala Arg Phe 805 810 815 US 6,534,300 B1 43 44

-continued Arg Val Glu Thr His Thr Gln Lys Thr Ile Gly Gly Asn Val Thr Lys 820 825 830 Glu Lys Pro Pro Ser Leu Ile Val Pro Leu Glu Ser Gln Met Thr Lys 835 840 845 Glu Lys Lys Ile Thr Gly Lys Glu Lys Glu Asn. Ser Arg Met Glu Glu 85 O 855 860 Asn Ala Glu Asn His Ile Gly Val Thr Glu Val Lieu Lleu Gly Arg Lys 865 870 875 88O Leu Gln His Tyr Thr Asp Ser Tyr Leu Gly Phe Leu Pro Trp Glu Lys 885 890 895 Lys Lys Tyr Phe Glin Asp Leu Lieu. Asp Glu Glu Glu Ser Lieu Lys Thr 9 OO 905 910 Glin Leu Ala Tyr Phe Thr Asp Ser Lys Asn Thr Gly Arg Glin Leu Lys 915 920 925

<210> SEQ ID NO 2 &2 11s LENGTH 328 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 2 Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Lieu. Asn. Ser 1 5 10 15 Lys Phe Gly Phe Thr Ser Arg Llys Val Pro Ala His Met Pro His Met 2O 25 30 Ile Asp Arg Ile Val Met Gln Glu Leu Glin Asp Met Phe Pro Glu Glu 35 40 45 Phe Asp Llys Thr Ser Phe His Lys Val Arg His Ser Glu Asp Met Glin 50 55 60 Phe Ala Phe Ser Tyr Phe Tyr Tyr Leu Met Ser Ala Val Glin Pro Leu 65 70 75 8O Asn Ile Ser Glin Val Phe Asp Glu Val Asp Thr Asp Gln Ser Gly Val 85 90 95 Leu Ser Asp Arg Glu Ile Arg Thr Lieu Ala Thr Arg Ile His Glu Lieu 100 105 110 Pro Leu Ser Lieu Glin Asp Lieu. Thr Gly Lieu Glu His Met Lieu. Ile Asn 115 120 125 Cys Ser Lys Met Leu Pro Ala Asp Ile Thr Glin Lieu. Asn. Asn. Ile Pro 130 135 1 4 0 Pro Thr Glin Glu Ser Tyr Tyr Asp Pro Asn Leu Pro Pro Val Thr Lys 145 15 O 155 160 Ser Lieu Val Thr Asn. Cys Lys Pro Val Thr Asp Lys Ile His Lys Ala 1.65 170 175 Tyr Lys Asp Lys Asn Lys Tyr Arg Phe Glu Ile Met Gly Glu Glu Glu 18O 185 190 Ile Ala Phe Lys Met Ile Arg Thr Asn Val Ser His Val Val Gly Glin 195 200 2O5 Leu Asp Asp Ile Arg Lys Asn Pro Arg Lys Phe Val Cys Lieu. Asn Asp 210 215 220 Asn. Ile Asp His Asn His Lys Asp Ala Glin Thr Val Lys Ala Val Lieu 225 230 235 240 Arg Asp Phe Tyr Glu Ser Met Phe Pro Ile Pro Ser Glin Phe Glu Leu 245 250 255 Pro Arg Glu Tyr Arg Asn Arg Phe Lieu. His Met His Glu Lieu Glin Glu 260 265 27 O US 6,534,300 B1 45 46

-continued Trp Arg Ala Tyr Arg Asp Lys Lieu Lys Phe Trp Thr His Cys Val Lieu 275 280 285

Ala Thr Lieu. Ile Met Phe Thir Ile Phe Ser Phe Phe Ala Glu Glin Leu 29 O 295 3OO Ile Ala Lieu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile His Lys Glu 305 310 315 320 Ala Ser Pro Asn Arg Ile Arg Val 325

<210> SEQ ID NO 3 &2 11s LENGTH 305 &212> TYPE PRT <213> ORGANISM: Homo sapiens &220s FEATURE <221 NAME/KEY: SIGNAL <222> LOCATION: (1) ... (24) <400 SEQUENCE: 3 Met Ala Ala Gly Lieu Ala Arg Lieu Lleu Lleu Lleu Lieu Gly Lieu Ser Ala 1 5 10 15 Gly Gly Pro Ala Pro Ala Gly Ala Ala Lys Met Lys Val Val Glu Glu 2O 25 30

Pro Asn Ala Phe Gly Val Asn Asn Pro Phe Leu Pro Glin Ala Ser Arg 35 40 45 Leu Glin Ala Lys Arg Asp Pro Ser Pro Wal Ser Gly Pro Wal His Lieu 50 55 60 Phe Arg Leu Ser Gly Lys Cys Phe Ser Leu Val Glu Ser Thr Tyr Lys 65 70 75 8O Tyr Glu Phe Cys Pro Phe His Asn Val Thr Gln His Glu Gln Thr Phe 85 90 95 Arg Trp Asn Ala Tyr Ser Gly Ile Leu Gly Ile Trp His Glu Trp Glu 100 105 110 Ile Ala Asn. Asn. Thir Phe Thr Gly Met Trp Met Arg Asp Gly Asp Ala 115 120 125 Cys Arg Ser Arg Ser Arg Glin Ser Lys Val Glu Lieu Ala Cys Gly Lys 130 135 1 4 0 Ser Asn Arg Leu Ala His Val Ser Glu Pro Ser Thr Cys Val Tyr Ala 145 15 O 155 160 Leu Thr Phe Glu Thr Pro Leu Val Cys His Pro His Ala Leu Leu Val 1.65 170 175 Tyr Pro Thr Leu Pro Glu Ala Leu Glin Arg Gln Trp Asp Glin Val Glu 18O 185 190 Glin Asp Leu Ala Asp Glu Lieu. Ile Thr Pro Glin Gly His Glu Lys Lieu 195 200 2O5 Leu Arg Thr Lieu Phe Glu Asp Ala Gly Tyr Lieu Lys Thr Pro Glu Glu 210 215 220 Asn Glu Pro Thr Glin Lieu Glu Gly Gly Pro Asp Ser Leu Gly Phe Glu 225 230 235 240 Thr Lieu Glu Asn. Cys Arg Lys Ala His Lys Glu Lieu Ser Lys Glu Ile 245 250 255 Lys Arg Lieu Lys Gly Lieu Lleu Thr Gln His Gly Ile Pro Tyr Thr Arg 260 265 27 O Pro Thr Glu Thir Ser Asn Leu Glu His Leu Gly His Glu Thr Pro Arg 275 280 285

US 6,534,300 B1 49 SO

-continued tgaatgccac cacaatacat tittaatctoa cgtttcaaaa tacaaacgat gaagagttca 20 40 aaatgcagat alacagtggag gtggacacaa gggagggacC aaaactgaat totacggcc.c 2100 agaagggitta cgaaaattta gttagtcc.ca talacactitct to Cagaggcg gaaatcctitt 216 O ttgaggatat toccaaagaa aaacgctitcc cgaagtttaa gag acatgat gttaactcaa 2220

Caaggaga.gc cCaggaagag gtgaaaattic ccctdgtaaa tattitcactic citt.ccaaaag 228O acgc.ccagtt gag totcaat accittggatt tgcaactgga acatggagac atcactittga 234. O aaggatacaa tttgtccaag tdagccttgc tgagatcatt totgatgaac to acago atg 24 OO citaaaataaa. aaatcaagct ataataa.cag atgaaacaaa tgacagtttg gtggctccac 2460 aggaaaaa.ca ggttcataaa agcatcttgc caaac agctt aggagtgtct gaaagattgc 252O agaggttgac tttitcctgca gtgagtgtaa aagtgaatgg tdatgaccag ggtoagaatc 258O caccc.ctgga cittggagacc acago aagat ttagagtgga aacticacacic Caaaaaa. C. Ca 264 O tagg.cggaaa tgtgacaaaa gaaaag.cccc catctotgat tgttccactg gaaagcc aga 27 OO tgacaaaaga aaagaaaatc acagggaaag aaaaagagaa cagtagaatg gaggaaaatg 276 O. citgaaaatca catagg.cgtt actgaagtgt tacttggaag aaagctgcag cattacacag 282O atagittacitt gggctttittg cCatgggaga aaaaaaagta tittccaagat cittctogacg 2880 aagaag agtc attgaagaca caattggcat actitcactga tag caaaaat actgggaggc 2.940 aactaaaaga tacatttgca gattccctca gatatgtaaa taaaatticta aatagdaagt ttggattoac atc.gcggaaa gtoccitgcto acatgccitca catgattgac cggattgtta 3060 tgcaagaact gcaagatatg titc.cctgaag aatttgacaa gacgtcattt cacaaagtgc 312 O gccattctga ggatatgcag tittgccttct cittatttitta ttatctoatg agtgcagtgc 318O agcc actaa tatatocticala gtotttgatg aagttgatac agatcaatct ggtgtc.ttgt 324 O citgacagaga aatcc.gaa.ca citggctacca gaattcacga actg.ccgtta agtttgcagg atttgacagg totggalacac atgctaataa attgctdaaa aatgctitcct gctgatato a 3360 cgcagotaaa taatatto.ca ccaactcagg aatcc tacta tgatcccaac citgccaccgg 342O toactaaaag totagtaaca aactgtaaac cagta actoga caaaatccac aaag catata 3480 aggacaaaaa caaatatagg tittgaaatca tgggagalaga agaaatcgct tittaaaatga 354. O titcgtaccaa cgtttctoat gtggttggCC agttggatga cataagaaaa aacco tagga 3600 agtttgtttg cctgaatgac aac attgacc acaat catala agatgcticag acagtgaagg 3660 citgttctoag gg acttctat gaatc catgt tocccatacc titcc.calatitt gaact gccala 372 O gagagtatcg aaaccotttc cittcatatgc atgagctgca ggaatggagg gcttatc gag 378 O. acaaattgaa gttittggacc cattgttgtac tag caacatt gattatgttt actatattoct 384 O cattttittgc tgagcagtta attgcactta agcggaagat attitcccaga aggaggatac 39 OO acaaagaa.gc tag toccaat c gaatcagag tatagaagat cittcatttga aalaccatcta 396 O cctdag catt tactgagcat tittaaaactic agctt cacag agatgtctitt gtgatgtgat 4020 gcttagcagt ttggcc.cgaa gaaggaaaat atccagtacc atgctgttitt gtggcatgaa 408 O tatagoccac tgacitaggaa ttatttalacc aac coactoga aaacttgtgt gtcgagcagc 414 O totgaactga titt tacttitt aaagaatttg citcatgg acc tgtcatccitt tittataaaaa. 4200 ggct cactga caa.gagacag citgttaattit cc cacagcaa. to attgcaga citalactititat 4260 taggagaa.gc citatgc.ca.gc tgggagtgat tgctaagagg citccagtctt tgcattccaa 4320 agccttittgc taaagttittg cacttitttitt tittitcatttic ccatttittaa. gtagttact a 4.380

US 6,534,300 B1 S3

-continued caaaggagat caaaaggctgaaaggtttgc ticacccagoa cgg catc.ccc tacac gaggc 840 ccacagaaac titccaacttg gag cacttgg gccac gagac gcc cagagcc aagtc.tc.ca.g 9 OO agcagotgcg gggtgaccca ggactg.cgtg ggagtttgttg accittgttggit gggagag cag 96.O aggtggacgc ggcc.gaga gC cctacagaga agctggctgg taggaccc.gc aggaccagot 1020 gaccaggctt gtgcto agag aag cagacaa aacaaagatt caaggttitta attaatticcic 1080 atactgataa aaataactcc atgaattctg. taaac cattg cataaatgct atagt gtaaa 1140 aaaatttaala caagttgttaa citttaaacag titc.gctacaa gtaaatgatt ataaatacta 1200 aaaaaaaaaa. aaaaaaaaa. 1219

SEQ ID NO 6 LENGTH 515 TYPE PRT ORGANISM Homo sapiens FEATURE: NAME/KEY: SIGNAL LOCATION: (1) . . (24) NAME/KEY: PROPEP LOCATION: (25) . . (49) <400 SEQUENCE: 6

Met Ala Thr Ser Thr Gly Arg Trp Lieu Lleu Lieu Arg Teu Ala Leu Phe 1 15

Gly Phe Telu Trp Glu Ala Ser Gly Gly Lieu. Asp Ser Gly Ala Ser Arg 25 30

Asp Asp Asp Telu Leu Lleu Pro Tyr Pro Arg Ala Arg Ala Arg Leu Pro 35 40 45

Arg Asp Thr Arg Val Arg Ala Gly Asn Arg Glu His Glu Ser Trp 50 55 60

Pro Pro Pro Pro Ala Thr Pro Gly Ala Gly Gly Teu Ala Wall Arg Thr 65 70 75 8O

Phe Wall Ser His Phe Arg Asp Arg Ala Val Ala Gly His Teu Thr Arg 85 90 95

Ala Wall Glu Pro Leu Arg Thr Phe Ser Val Leu Glu Pro Gly Gly Pro 100 105 110

Gly Gly Cys Ala Ala Arg Arg Arg Ala Thr Val Glu Glu Thr Ala Arg 115 120 125

Ala Ala Asp Arg Val Ala Glin Asn Gly Gly Phe Phe Arg Met Asn 130 135 1 4 0

Ser Gly Glu Leu Gly Asn Val Val Ser Asp Glu Arg Arg Wal Ser 145 15 O 155 160

Ser Ser Gly Gly Leu Glin Asn Ala Glin Phe Gly Ile Arg Arg Asp Gly 1.65 170 175

Thr Telu Wall Thr Gly Tyr Lieu Ser Glu Glu Glu Wall Teu Asp Thr Glu 18O 185 190

Asn Pro Phe Wall Gln Leu Leu Ser Gly Val Val Trp Teu Ile Arg Asn 195 200

Gly Ser Ile Ile Asn. Glu Ser Glin Ala Thr Glu Asp Glu Thr 210 215 220

Glin Glu Thr Gly Ser Phe Ser Lys Phe Val Asn Wall Ile Ser Ala Arg 225 230 235 240

Thr Ala Ile Gly His Asp Arg Lys Gly Glin Lieu Wall Teu Phe His Ala 245 250 255

Asp Gly His Thr Glu Glin Arg Gly Ile Asn Lieu Trp Glu Met Ala Glu 260 265 27 O

US 6,534,300 B1 59 60

-continued Pro Leu Ser Lieu Glin Asp Lieu. Thr Gly Lieu Glu His Met Lieu. Ile Asn 115 120 125 Cys Ser Lys Met Leu Pro Ala Asn. Ile Thr Glin Lieu. Asn. Asn. Ile Pro 130 135 1 4 0 Pro Thr Glin Glu Ala Tyr Tyr Asp Pro Asn Leu Pro Pro Val Thr Lys 145 15 O 155 160 Ser Lieu Val Thr Asn. Cys Lys Pro Val Thr Asp Lys Ile His Lys Ala 1.65 170 175 Tyr Lys Asp Lys Asn Lys Tyr Arg Phe Glu Ile Met Gly Glu Glu Glu 18O 185 190 Ile Ala Phe Lys Met Ile Arg Thr Asn Val Ser His Val Val Gly Glin 195 200 2O5 Leu Asp Asp Ile Arg Lys Asn Pro Arg Lys Phe Val Cys Lieu. Asn Asp 210 215 220 Asn. Ile Asp His Asn His Lys Asp Ala Arg Thr Val Lys Ala Val Lieu 225 230 235 240 Arg Asp Phe Tyr Glu Ser Met Phe Pro Ile Pro Ser Glin Phe Glu Leu 245 250 255 Pro Arg Glu Tyr Arg Asn Arg Phe Lieu. His Met His Glu Lieu Glin Glu 260 265 27 O Trp Arg Ala Tyr Arg Asp Lys Lieu Lys Phe Trp Thr His Cys Val Lieu 275 280 285

Ala Thr Lieu. Ile Ile Phe Thir Ile Phe Ser Phe Phe Ala Glu Glin Ile 29 O 295 3OO Ile Ala Lieu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile His Lys Glu 305 310 315 32O Ala Ser Pro Asp Arg Ile Arg Val 325

<210 SEQ ID NO 9 &2 11s LENGTH 307 &212> TYPE PRT <213> ORGANISM: Mus musculus

<400 SEQUENCE: 9 Met Ala Gly Arg Lieu Ala Gly Phe Lieu Met Leu Lieu Gly Lieu Ala Ser 1 5 10 15 Glin Gly Pro Ala Pro Ala Cys Ala Gly Lys Met Lys Val Val Glu Glu 2O 25 30 Pro Asn Thr Phe Gly Leu Asn Asn Pro Phe Leu Pro Glin Ala Ser Arg 35 40 45 Leu Gln Pro Lys Arg Glu Pro Ser Ala Wal Ser Gly Pro Lieu. His Lieu 50 55 60 Phe Arg Lieu Ala Gly Lys Cys Phe Ser Lieu Val Glu Ser Thr Tyr Lys 65 70 75 8O Tyr Glu Phe Cys Pro Phe His Asn Val Thr Gln His Glu Gln Thr Phe 85 90 95 Arg Trp Asn Ala Tyr Ser Gly Ile Leu Gly Ile Trp His Glu Trp Glu 100 105 110 Ile Ile Asin Asn Thr Phe Lys Gly Met Trp Met Thr Asp Gly Asp Ser 115 120 125 Cys His Ser Arg Ser Arg Glin Ser Lys Val Glu Lieu. Thir Cys Gly Lys 130 135 1 4 0 Ile Asin Arg Leu Ala His Val Ser Glu Pro Ser Thr Cys Val Tyr Ala 145 15 O 155 160

US 6,534,300 B1 63 64

-continued tacagoggga to cittggcat citggcatgag toggaaatca tdaacaatac cittcaagggc 200 atgtggatga citgatgggga citcct gccac toccggagcc ggcagagcaa gotggagctic 260 acct gtggaa agatcaa.ccg actggcc.cac gtgtctgagc caa.gcacct g totcitat gca 320 ttgacattcg agaccoctot tatttgccat coccactcitt tgttagtgta tocaactctg to agaa.gc.cc tdcago agcc cittggaccag gtggaac agg acctggcaga tigaactogatc 4 40 acaccacagg gctato agaa gttgctaagg gtactttittg aggatgctogg citacttaaag 5 OO gtoccaggag aaa.cccatcc cacco agctg gcaggaggitt cCaagggcct ggggcttgag 560 actctggaca act gtagaaa gocacatgca gagctgtcac aggaggtaca aag actogacg agtctgctgc aacago atgg aatcc.cccac acticagocca caggtoagtic tocctg.ccct 680 gg to agctgc cagocacticc ggggcctgca gcactggggc agatctittat tigctacccat 740 totggcagaa accact cact citcago acct g g g to agcag citc.cccatag gtgcaatcgc 800 agcagagcat citgcggagtg accoaggact acgtgggaac atcct gtgag caaggtogcc 860 acgaagaata gaaatatoct gagctittgag tdtcc tittca cagagtgaac aaaactogtg 920 tggtgtagac acggcttctt ttggcatatt citagatcaga cagtgtcact gacaaacaag agg gacct gc tiggccago: ct ttgttgttgcc caaagatcca gacaaaataa agattcaaag 20 40 ttittaattaa aaaaaaaaaa aaaggaattic 2070

SEQ ID NO 11 LENGTH 113 TYPE PRT ORGANISM Rattus rattus

<400 SEQUENCE: 11 Phe Pro Pro Thr Phe Lys Glu Thir Ser Glin Leu Lys Thr Lys Lieu Pro 1 5 10 15 Glu Asn Lieu Ser Ser Lys Ile Lys Lieu Lieu Glin Leu Tyr Ser Glu Ala 2O 25 30 Ser Val Ala Lieu Lleu Lys Lieu. Asn. Asn Pro Lys Gly Phe Pro Glu Leu 35 40 Asn Lys Glin Thr Lys Lys Asn Met Ser Ile Ser Gly Lys Glu Lieu Ala 50 55 60

Ile Ser Pro Ala Tyr Lieu Lleu Trp Asp Leu Ser Ala Ile Ser Glin Ser 65 70 75 8O Lys Glin Asp Glu Asp Wal Ser Ala Ser Arg Phe Glu Asp Asn. Glu Glu 85 90 95

Leu Arg Tyr Ser Leu Arg Ser Ile Glu Arg His Asp Ser Met Ser Pro 100 105 110

Teu

SEQ ID NO 12 LENGTH 460 TYPE DNA ORGANISM Rattus rattus

<400 SEQUENCE: 12 attcccacca acattcaagg agacgagtica gctgaag aca aaact gccag aaaatctttc 60 ttctaaaata aaactgttgc agctdtactic ggaggcc agc gtogctcittctgaaattgaa 120 taac cocaaa gqtttcc.ccg agctdaacaa goagaccaag aagaacatga gcatcagtgg 18O gaaggaactg gcc atcagoc citgccitat cit gctgtgggac citgagcgc.ca totago cagtic 240 US 6,534,300 B1 65

-continued caag caggat galagatgtgt citgccago.cg citt.cgaggat aac galaga.gc tigagg tactic 3OO actgagat.ct atc.gagagac atgattcc at gagtoctitta toga attctgg ccatatottc 360 aatcatgatc. tcagtagitat toctotgaaa tagg cacacat ttittctaatg agaacttgaa 420 atgtaaatat tigtgtttgttg citgitaaattt tatgtatttc 460

<210> SEQ ID NO 13 &2 11s LENGTH 502 &212> TYPE PRT <213> ORGANISM: Drosophila melanogaster <400 SEQUENCE: 13 Gly Thr Arg Arg Phe Asp Asp Lys Asn. Glu Lieu Arg Tyr Ser Lieu Arg 1 5 10 15 Ser Leu Glu Lys His Ala Ala Trp Ile Arg His Val Tyr Ile Val Thr 2O 25 30 Asn Gly Glin Ile Pro Ser Trp Lieu. Asp Leu Ser Tyr Glu Arg Val Thr 35 40 45 Val Val Pro His Glu Val Leu Ala Pro Asp Pro Asp Gln Leu Pro Thr 50 55 60 Phe Ser Ser Ser Ala Ile Glu Thr Phe Leu. His Arg Ile Pro Llys Leu 65 70 75 8O Ser Lys Arg Phe Leu Tyr Lieu. Asn Asp Asp Ile Phe Leu Gly Ala Pro 85 90 95 Leu Tyr Pro Glu Asp Leu Tyr Thr Glu Ala Glu Gly Val Arg Val Tyr 100 105 110 Gln Ala Trp Met Val Pro Gly Cys Ala Leu Asp Cys Pro Trp Thr Tyr 115 120 125 Ile Gly Asp Gly Ala Cys Asp Arg His Cys Asn. Ile Asp Ala Cys Glin 130 135 1 4 0 Phe Asp Gly Gly Asp Cys Ser Glu Thr Gly Pro Ala Ser Asp Ala His 145 15 O 155 160 Val Ile Pro Pro Ser Lys Glu Val Leu Glu Val Glin Pro Ala Ala Val 1.65 170 175 Pro Glin Ser Arg Val His Arg Phe Pro Glin Met Gly Leu Gln Lys Leu 18O 185 190 Phe Arg Arg Ser Ser Ala Asn. Phe Lys Asp Wal Met Arg His Arg Asn 195 200 2O5 Val Ser Thr Lieu Lys Glu Lieu Arg Arg Ile Val Glu Arg Phe Asn Lys 210 215 220 Ala Lys Lieu Met Ser Lieu. Asn. Pro Glu Lieu Glu Thir Ser Ser Ser Glu 225 230 235 240 Pro Glin Thir Thr Glin Arg His Gly Lieu Arg Lys Glu Asp Phe Lys Ser 245 250 255 Ser Thr Asp Ile Tyr Ser His Ser Leu Ile Ala Thr Asn Met Leu Leu 260 265 27 O Asn Arg Ala Tyr Gly Phe Lys Ala Arg His Val Lieu Ala His Val Gly 275 280 285 Phe Lieu. Ile Asp Lys Asp Ile Val Glu Ala Met Glin Arg Arg Phe His 29 O 295 3OO Glin Glin Ile Leu Asp Thr Ala His Glin Arg Phe Arg Ala Pro Thr Asp 305 310 315 320 Leu Glin Tyr Ala Phe Ala Tyr Tyr Ser Phe Leu Met Ser Glu Thr Lys 325 330 335 US 6,534,300 B1 67 68

-contin ued Wal Met Ser Wall Glu Glu Ile Phe Asp Glu Phe Asp Thr Asp Gly Ser 340 345 350

Ala Thr Trp Ser Asp Arg Glu Val Arg Thr Phe Lieu. Thr Arg Ile Tyr 355 360 365

Gln Pro Pro Telu Asp Trp Ser Ala Met Arg Tyr Phe Glu Glu Wal Wall 370 375 38O

Glin Asn. Cys Thr Arg Asn Lieu Gly Met His Leu Lys Val Asp Thr Wall 385 390 395 400

Glu. His Ser Thr Teu Val Tyr Glu Arg Tyr Glu Asp Ser Asn Leu Pro 405 410 415

Thir Ile Thr Arg Asp Leu Wal Wall Arg Cys Pro Leu Lleu Ala Glu Ala 420 425 430

Leu Ala Ala Asn Phe Ala Val Arg Pro Llys Tyr Asn Phe His Wal Ser 435 4 40 4 45

Pro Lys Arg Thr Ser His Ser Asn Phe Met Met Leu. Thir Ser Asn Lieu 450 455 460

Thr Glu Wall Wall Glu Ser Lieu. Asp Arg Lieu Arg Arg Asn Pro 465 470 475 480

Phe Asn. Cys Ile Asn Asp Asn Lieu Asp Ala Asn Arg Gly Glu Asp Asn 485 490 495

Glu Asp Gly Ala Pro Ser 5 OO

<210> SEQ ID NO 14 &2 11s LENGTH 9792 &212> TYPE DNA <213> ORGANISM: Mus musculus

<400 SEQUENCE: 14

Caggct cqgg actitactata acacaggaca cittgtcacct gaaagcttga gtoagtcagt 60 tattatggto tgtgtgtgag atacaagtgg gtgcataggc agtggtgcac acatgtagat 120 cag actittct acagocaatt citcttctitcc to citctic cat gggttcaggg tottcatc.tc. 18O aggttgcaca gcq agttcat titatgtgctg tgccatctog ccagtcgttc citatatocta 240 gaggaaaact agtttcttct g g to aagagg aggaaagagt ggagacct gt cattctaaga tacccaaaac agggcCaggit toggggacCtg tgcctittaat cc catcactit ggggattagg 360 tagaa.gcaag aggctotaga ccagtctaca cactgaattit caa.gc.cago c tacctataaa. 420 to agaga.ccc tgcttcaaaa ataaaattaa acaaaaacga agataalacca agctacccala 480 aacacaagag ttaatccagt cagacaggto tag caaatgc taggatgaaa ggtgtgcacc 540 accacgagtg ggctgcaagc citctotctot citcticitcticit citcticitctict citcgtttgtt 600 ttgtttittcg agacaaggitt totctgtgta gcc citggctg to citggaact cactctgtag 660 acCaggctgg cctcgagctt cactcittaaa agttcctcitt ccitcc ticcitc. catc.tttitcc. 720 to citc.ttacc cccitaggcto cittittcctct tottgttctitt cagataaagt citcaagtagt ccagacitggit citcaaactaa citaactag co aagaatagoc alacct cittaa. cittcc.gattc 840 toctgccitct gctgaatgct ggggttgttgg CgtgggCCaC cacttctggit ttgttgcaa.ca 9 OO cagaaggaac tagggctitta agcac gagaa gcaagttctg tacagacitta cacaggcc.ca 96.O gcatctgttc ttgcaattitt citgitaagttt gacataatat gagaataaaa agctatotat 1020 citcc ct tcca gccttaccct citctgatgga attic gaatgc gtaatcaaag cacccaa.ca.g 1080 cctggcct ga aatcacgtgg ggcaa.gcc.ca Cgtgaccgga gcaccaatcc aatatgg.cgg 1140

US 6,534,300 B1 77 78

-continued

Asn Thr Thr Glu Pro Thr Lys Ser Glu Lys Glin Teu Glu 115 120 125

Teu Telu Thr His Ile Lys Wall Pro Met Teu Wall Teu Asp Pro Ala 130 135 1 4 0

Teu Pro Ala Thr Ile Thr Teu Asp Telu Pro Thr Teu Pro Ser 145 15 O 155 160

Phe His Ala Ser Ser Asp Met Phe Asn Wall Ala Lys Pro Asn Pro 1.65 170 175

Ser Thr Asn Wall Pro Wall Wall Wall Phe Asp Thr Thr Asp Wall Glu 18O 185 190

Asp Ala His Ala Gly Pro Phe Lys Gly Gly Glin Glin Thr Asp Wall Trp 195 200

Arg Ala Telu Thr Thr Asp Asp Ala Pro Gly Teu Wall Telu Ile 210 215 220

Glin Gly Telu Ala Phe Teu Ser Gly Phe Pro Pro Thr Phe Glu Thr 225 230 235 240

Ser Glin Telu Thr Teu Pro Arg Lys Ala Phe Pro Teu Lys Ile 245 250 255

Telu Telu Arg Teu Ser Glu Ala Ser Wall Ala Teu Teu Lys Telu 260 265 27 O

Asn Asn Pro Gly Glin Glu Telu Asn Glin Thr Lys Asn 275 280 285

Met Thr Ile Asp Gly Glu Telu Thr Ile Ser Pro Ala Telu Telu 29 O 295

Trp Asp Telu Ser Ala Ile Ser Glin Ser Glin Asp Glu Asp Ala Ser 305 310 315

Ala Ser Arg Phe Glu Asp Asn Glu Glu Telu Arg Ser Teu Arg Ser 325 330 335

Ile Glu Arg His Ala Pro Trp Wall Arg Asn Ile Phe Ile Wall Thr Asn 340 345 350

Gly Glin Ile Pro Ser Trp Teu Asn Telu Asp Asn Pro Arg Wall Thr Ile 355 360 365

Wall Thr His Glin Asp Ile Phe Glin Asn Telu Ser His Teu Pro Thr Phe 370 375

Ser Ser Pro Ala Ile Glu Ser His Ile His Arg Ile Glu Telu Ser 385 390 395 400

Glin Phe Ile Tyr Teu Asn Asp Asp Wall Met Phe Gly Tys Asp Wall 405 410 415

Trp Pro Asp Asp Phe Ser His Ser Gly Glin Wall Tyr Telu 420 425 430

Thr Trp Pro Wall Pro Asn Ala Glu Gly Pro Gly Ser Trp Ile 435 4 40 4 45

Asp Gly Asp Lys Ala Asn Thr Ser Pro Trp 450 455 460

Asp Gly Gly Asn Ser Gly Asn Thr Ala Gly Asn Arg Phe Wall Ala 465 470 475 480

Arg Gly Gly Gly Thr Gly Asn Ile Gly Ala Gly Glin His Trp Glin Phe 485 490 495

Gly Gly Gly Ile Asn Thr Ile Ser Tyr Asn Glin Gly Cys Ala Asn 5 OO 505 510

Ser Trp Telu Ala Asp Phe Cys Asp Glin Ala Asn Wall Telu Ser 515 52O 525 US 6,534,300 B1 79 80

-continued Cys Gly Phe Asp Ala Gly Asp Cys Gly Glin Asp His Phe His Glu Lieu 530 535 540 Tyr Lys Val Thr Leu Leu Pro Asn Gln Thr His Tyr Val Val Pro Lys 545 550 555 560 Gly Glu Tyr Lieu Ser Tyr Phe Ser Phe Ala Asn. Ile Ala Arg Lys Arg 565 570 575 Ile Glu Gly Thr Tyr Ser Asp Asn Pro Ile Ile Arg His Ala Ser Ile 58O 585 59 O Ala Asn Lys Trp Lys Thr Lieu. His Lieu. Ile Met Pro Gly Gly Met Asn 595 600 605 Ala Thir Thir Ile Tyr Phe Asn Lieu. Thir Lieu Glin Asn Ala Asn Asp Glu 610 615 62O Glu Phe Lys Ile Glin Ile Ala Val Glu Val Asp Thr Arg Glu Ala Pro 625 630 635 640 Lys Leu Asn Ser Thr Thr Gln Lys Ala Tyr Glu Ser Leu Val Ser Pro 645 650 655 Val Thr Pro Leu Pro Glin Ala Asp Val Pro Phe Glu Asp Val Pro Lys 660 665 670 Glu Lys Arg Phe Pro Lys Ile Arg Arg His Asp Wall Asn Ala Thr Gly 675 680 685 Arg Phe Glin Glu Glu Wall Lys Ile Pro Arg Val Asn. Ile Ser Lieu Lieu 69 O. 695 7 OO Pro Lys Glu Ala Glin Val Arg Lieu Ser Asn Lieu. Asp Lieu Glin Leu Glu 705 710 715 720 Arg Gly Asp Ile Thr Lieu Lys Gly Tyr Asn Lieu Ser Lys Ser Ala Lieu 725 730 735 Leu Arg Ser Phe Leu Gly Asn. Ser Lieu. Asp Thr Lys Ile Llys Pro Glin 740 745 750 Ala Arg Thr Asp Glu Thir Lys Gly Asn Lieu Glu Val Pro Glin Glu Asn 755 760 765 Pro Ser His Arg Arg Pro His Gly Phe Ala Gly Glu His Arg Ser Glu 770 775 78O Arg Trp Thr Ala Pro Ala Glu Thr Val Thr Val Lys Gly Arg Asp His 785 790 795 8OO

Ala Lieu. Asn Pro Pro Pro Val Lieu Glu Thir Asn Ala Arg Lieu Ala Glin 805 810 815

Pro Thr Leu Gly Val Thr Val Ser Lys Glu Asn Leu Ser Pro Leu Ile 820 825 830 Val Pro Pro Glu Ser His Leu Pro Lys Glu Glu Glu Ser Asp Arg Ala 835 840 845 Glu Gly Asn Ala Val Pro Wall Lys Glu Lieu Val Pro Gly Arg Arg Lieu 85 O 855 860 Gln Glin Asn Tyr Pro Gly Phe Leu Pro Trp Glu Lys Lys Lys Tyr Phe 865 870 875 88O Glin Asp Lieu Lieu. Asp Glu Glu Glu Ser Lieu Lys Thr Glin Leu Ala Tyr 885 890 895 Phe Thr Asp Arg Lys His Thr Gly Arg Glin Leu Lys 9 OO 905

<210> SEQ ID NO 16 &2 11s LENGTH 5229 &212> TYPE DNA <213> ORGANISM: Mus musculus

US 6,534,300 B1 89 90

-continued <210 SEQ ID NO 19 &2 11s LENGTH 492 &212> TYPE PRT <213> ORGANISM: Mus musculus

<400 SEQUENCE: 19 Val Ser Arg Asp Asp Asp Leu Lleu Lieu Pro Tyr Pro Leu Ala Arg Arg 1 5 10 15 Arg Pro Ser Arg Asp Cys Ala Arg Val Arg Ser Gly Ser Pro Glu Glin 2O 25 30 Glu Ser Trp Pro Pro Pro Pro Leu Ala Thr His Glu Pro Arg Ala Pro 35 40 45 Ser His His Ala Ala Val Arg Thr Phe Val Ser His Phe Glu Gly Arg 50 55 60 Ala Val Ala Gly His Lieu. Thr Arg Val Ala Asp Pro Leu Arg Thr Phe 65 70 75 8O Ser Val Lieu Glu Pro Gly Gly Ala Gly Gly Cys Gly Gly Arg Ser Ala 85 90 95 Ala Ala Thr Val Glu Asp Thr Ala Val Arg Ala Gly Cys Arg Ile Ala 100 105 110 Glin Asn Gly Gly Phe Phe Arg Met Ser Thr Gly Glu Cys Lieu Gly Asn 115 120 125 Val Val Ser Asp Gly Arg Lieu Val Ser Ser Ser Gly Gly Lieu Glin Asn 130 135 1 4 0 Ala Glin Phe Gly Ile Arg Arg Asp Gly Thr Ile Val Thr Gly Ser Cys 145 15 O 155 160 Leu Glu Glu Glu Val Leu Asp Pro Val Asn Pro Phe Val Glin Leu Leu 1.65 170 175 Ser Gly Val Val Trp Lieu. Ile Arg Asn Gly Asn. Ile Tyr Ile Asn. Glu 18O 185 190 Ser Glin Ala Ile Glu Cys Asp Glu Thr Glin Glu Thr Gly Ser Phe Ser 195 200 2O5 Lys Phe Val Asn Val Met Ser Ala Arg Thr Ala Val Gly His Asp Arg 210 215 220 Glu Gly Glin Lieu. Ile Leu Phe His Ala Asp Gly Glin Thr Glu Glin Arg 225 230 235 240 Gly Lieu. Asn Lieu Trp Glu Met Ala Glu Phe Leu Arg Glin Glin Asp Wal 245 250 255 Val Asn Ala Ile Asn Lieu. Asp Gly Gly Gly Ser Ala Thr Phe Val Lieu 260 265 27 O Asn Gly Thr Lieu Ala Ser Tyr Pro Ser Asp His Cys Glin Asp Asn Met 275 280 285 Trp Arg Cys Pro Arg Glin Val Ser Thr Val Val Cys Val His Glu Pro 29 O 295 3OO Arg Cys Glin Pro Pro Asp Cys Ser Gly His Gly Thr Cys Wall Asp Gly 305 310 315 320 His Cys Glu Cys Thr Ser His Phe Trp Arg Gly Glu Ala Cys Ser Glu 325 330 335 Leu Asp Cys Gly Pro Ser Asn. Cys Ser Gln His Gly Lieu. Cys Thr Ala 340 345 350 Gly Cys His Cys Asp Ala Gly Trp Thr Gly Ser Asn. Cys Ser Glu Glu 355 360 365 Cys Pro Leu Gly Trp Tyr Gly Pro Gly Cys Glin Arg Pro Cys Glin Cys 370 375 38O US 6,534,300 B1 91 92

-contin ued Glu. His Glin Phe Cys Asp Pro Gln Thr Gly Asn. Cys Ser Ile Ser 385 390 395 400

Glin Val Arg Glin Cys Leu Glin Pro Thr Glu Ala Thr Pro Arg Ala Gly 405 410 415

Glu Lieu Ala Ser Phe Thr Arg Thr Thr Trp Leu Ala Lieu. Thr Leu. Thr 420 425 430

Leu. Ile Phe Telu Teu Lieu. Ile Ser Thr Gly Val Asn Wal Ser Leu Phe 435 4 40 4 45

Leu Gly Ser Arg Ala Glu Arg Asn Arg His Lieu Asp Gly Asp Tyr Val 450 455 460

Tyr His Pro Telu Glin Glu Wall Asn Gly Glu Ala Lieu. Thir Ala Glu Lys 465 470 475 480

Glu. His Met Glu Glu Thir Ser Asn Pro Phe Lys Asp 485 490

<210> SEQ ID NO 20 &2 11s LENGTH 37.83 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 20 gccaccatgg ggttcaagct cittgcagaga caaaccitata cctg.cctgtc ccacaggitat 60 gggctotacg tgtgcttctt go.gcgtogtt gtoac catcg totcc.gc.citt ccagttcgga 120 gaggtggttc tggaatggag cc.gagatcaa taccatgttt tgtttgattc citatagagac 18O aatattgctg gaaagticcitt to agaatcgg citttgttctg.c ccatgcc gat tgacgttgtt 240 tacaccitggg tgaatggcac agatcttgaa citactgaagg aactacagoa ggtoagagaa

Cagatggagg aggagcagaa agcaatgaga gaaatcc ttg ggaaaaacac aacggaacct 360 actaagaaga gtgagaag ca gttagagtgt ttgctaacac actgcattaa ggit gccaatg 420 cittgtc.ctgg accoagcc cit gcc agccaac atcaccotga agg acct go c atctott tat 480 cc titc.tttitc. attctgccag tdacatttitc aatgttgcaa. a C Caaaaaa. cc cittctacc 540 aatgtc.tcag ttgttgttitt tdacagtact aag gatgttg aagatgcc.ca citctggactg 600 cittaaaggaa atagoagaca gacagtatgg aggggctact tgacaacaga taaagaagtic 660 cctggattag tgctaatgca agatttggct titcctgagtg gattitccacc aac attcaag 720 gaaacaaatc aactaaaaac aaaattgc.ca gaaaatctitt cctctaaagt caaactgttg cagttgtatt cagaggccag togtag cqctt citaaaactoga ataa.ccc.cala ggattittcaa 840 gaattgaata agcaaactaa gaagaacatg accattgatg gaaaagaact gaccatalagt 9 OO cctgcatatt tattatggga totgagc.gc.c atcago cagt citaag cagga tgaagacatc 96.O totgccagtc gttittgaaga taacgaagaa citgaggtact cattgcgatc tat cq agagg O20 catgcaccat gggttcggaa tattittcatt gtocaccaacg ggcagattoc atcctggctg aaccttgaca atccitcgagt gacaatagta acacaccagg atgtttittcg aaatttgagc 14 O cacttgccta cctittagttc acctgctatt gaaagtcacg ttcatc.gcat C galagggctg 200 toccagaagt ttatttacct aaatgatgat gtoatgtttg ggalaggatgt citggccagat 260 gattitttaca gtoacticcaa aggcc agaag gtttatttga catggcc tot gccaaactot 320 gcc.gagggct gcc caggttc. citygattaag gatggctatt gtgacaaggc ttgtaataat to agcctg.cg attgggatgg toggggattgC totggaaa.ca gtggagggag togctatatt 4 40 gCaggaggtg gaggtactgg gagtattgga gttggacago cctgg cagtt tggtggagga 5 OO US 6,534,300 B1 93 94

-continued ataaac agtg totcttactg taatcaggga tgtgc gaatt cctggctic go tgataagttc 1560 tgtgaccalag catgcaatgt cittgtc.ctgt gggtttgatg citggc gactg tgggcaagat 1620 catttitcatg aattgtataa agtgatccitt citcc.caaacc agacitcacta tattatto.ca 1680 aaaggtgaat gcctg.ccitta tittcagottt gCagaagtag ccaaaag agg agttgaaggt 1740 gcctatagtg acaatccaat aatticgacat gcttctattg ccaacaagtg gaaaaccatc 1800 caccitcatala tgcacagtgg aatgaatgcc accacaatac attittaatct cacgtttcaa 1860 aatacaaacg atgaag agtt caaaatgcag atalacagtgg aggtegaCaC aagggaggga 1920 ccaaaactga attctacggc C Cagaagggit tacgaaaatt tagittagtoc catalacactit 1980 cittccagagg cggaaatcct ttittgaggat attcc caaag aaaaacgctt ccc.gaagttt 20 40 aagaga catg atgttaactc aacaaggaga gccCaggaag aggtgaaaat toccctggta 2100 aatattitcac toctitccaaa. agacgcc.cag ttgagtctoa ataccttgga tittgcaactg 216 O galacatggag acatcactitt gaaaggatac aatttgtc.ca agt cago citt gct gagatca 2220 tittctgatga acticacagoa tgctaaaata aaaaatcaag citataataac agatgaaa.ca 228O aatgacagtt tggtggcticc acaggaaaaa caggttcata aaag catctt gccaaac agc 234. O ttaggagtgt citgaaagatt gCagaggttg acttittcctg cagtgagtgt aaaagtgaat 24 OO gg to atgacc agggtoagaa tocaccoct9. gacittggaga ccacagdaag atttagagtg 2460 gaaact caca CCC aaaaaac Catagg.cgga aatgttgacaa aagaaaag.cc cccatctotg 252O attgttccac tggaaag.cca gatgacaaaa gaaaagaaaa to acagggaa agaaaaagag 258O aacagtagaa tggaggaaaa tgctgaaaat cacatagg.cg ttactgaagt gttacittgga 264 O agaaagctgc agcattacac agatagittac ttgggcttitt tgc catggga gaaaaaaaag 27 OO tattitcctag atcttctoga C galagaagag to attgaaga cacaattggc a tactitcact 276 O. gatago aaga atactgggag gcaactaaaa gatacatttg cag attccot cagatatgta 282O aataaaattic taaatagoaa gtttggattc acatcgcgga aagtc.cct gc tdacatgcct 2880 cacatgattg accggattgt tatgcaagaa citgcaagata tgttccctga agaatttgac 2.940 aagacgtoat ttcacaaagt gcgc.cattct gaggatatgc agtttgccitt citc.ttattitt tattatctoa tgagtgcagt gcago cactg aatatatoctic aagtc.tttga tgaagttgat 3060 acagat caat citggtgtctt gtotgacaga gaaatcc gaa cactggctac cagaattcac 312 O gaactg.ccgt. taagtttgca ggatttgaca ggtotggaac acatgctaat aaattgctoa 318O aaaatgctitc citgctgatat cacgcagota aataatatto caccalactica ggaatccitac 324 O tatgatccca acctg.ccacc ggtoactaaa agtictagtaa caaactgtaa accagtaact gacaaaatcc acaaag cata taaggacaaa aacaaatata ggitttgaaat Catgggagaa 3360 gaagaaatcg cittittaaaat gattcgtacc aacgtttcto atgtggttgg ccagttggat 342O gacataagaa aaaaccotag gaagtttgtt tgcctgaatg acaac attga ccacaatcat 3480 aaagatgctc agacagtgaa ggctgttcto agg gacittct atgaatccat gttcc.ccata 354. O cct tcc caat ttgaactgcc aagagagtat cgaalacc gtt toctitcaitat gcatgagctg 3600

Caggaatgga gggcttatcg agacaaattg aagttittgga cccattgttgt actagoaa.ca 3660 ttgattatgt titactatatt citcattittitt gctgagcagt taattgcact taag.cggaag 372 O a tattit.ccca gaaggaggat acacaaagaa gctagtc.cca atc gaatcag agtatagaag 378 O. atc 3783

US 6,534,300 B1 101 102

-continued SEQ ID NO 27 LENGTH 2.8 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 27 Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Lieu. Asn. Ser 1 5 10 15 Lys Phe Gly Phe Thr Ser Arg Llys Val Pro Ala His 2O 25

SEQ ID NO 28 LENGTH 21 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 28 Ala Lys Met Lys Val Val Glu Glu Pro Asn. Thr Phe Gly Lieu. Asn. Asn 1 5 10 15

Pro Phe Leu Pro Glin 2O

SEQ ID NO 29 LENGTH 5 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 29 Ile Lieu. Asn. Ser Lys 1 5

SEQ ID NO 30 LENGTH 5 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 30 Thr Ser Phe His Lys 1

SEQ ID NO 31 LENGTH 6 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 31 Phe Gly Phe Thr Ser Arg 1 5

SEQ ID NO 32 LENGTH 12 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 32 Ser Lieu Val Thr Asn. Cys Lys Pro Val Thr Asp Lys 1 5 10

SEQ ID NO 33 LENGTH 12 TYPE PRT ORGANISM Bos taurus US 6,534,300 B1 103 104

-continued SEQUENCE: 33 Leu Ala His Val Ser Glu Pro Ser Thr Cys Val Tyr 1 5 10

SEQ ID NO 34 LENGTH 13 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 34 Asn Asn Pro Phe Leu Pro Gln Thr Ser Arg Leu Gln Pro 1 5 10

SEQ ID NO 35 LENGTH 17 TYPE PRT ORGANISM Bos taurus FEATURE: NAME/KEY: misc feature LOCATION: (8) ... (8) OTHER INFORMATION Xaa is any amino acid NAME/KEY: misc feature LOCATION: (10) . . (10) OTHER INFORMATION Xaa is any amino acid NAME/KEY: misc feature LOCATION: (13) . . (13) OTHER INFORMATION Xaa is any amino acid NAME/KEY: misc feature LOCATION: (15) . . (15) OTHER INFORMATION Xaa is any amino acid

<400 SEQUENCE: 35 Val Pro Met Leu Val Leu Asp Xaa Ala Xaa Pro Thr Xaa Wal Xala Leu 5 10 15

SEQ ID NO 36 LENGTH 22 TYPE PRT ORGANISM Bos taurus

<400 SEQUENCE: 36 Glu Leu Pro Ser Leu Tyr Pro Ser Phe Leu Ser Ala Ser Asp Val Phe 1 5 10 15 Asn Val Ala Lys Pro Lys

SEQ ID NO 37 LENGTH 25 TYPE DNA ORGANISM: Artificial/Unknown FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 37 gCgaagatga aggtogtgga ggacC

SEQ ID NO 38 LENGTH 24 TYPE DNA ORGANISM: Artificial/Unknown FEATURE: NAME/KEY: misc feature LOCATION: () . . () US 6,534,300 B1 105 106

-continued <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 38 tgcagagaca gacctatacc tocc 24

<210 SEQ ID NO 39 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial/Unknown &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: () . . () <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 39 actcaccitct cogaactgga aag 23

<210> SEQ ID NO 40 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial/Unknown &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: () . . () <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 40 citagccacca toggggttcaa got cittgca 29

<210> SEQ ID NO 41 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial/Unknown &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: () . . () <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 41 agagcttgaa ccc catggtg g 21

<210> SEQ ID NO 42 &2 11s LENGTH 60 &212> TYPE DNA <213> ORGANISM: Artificial/Unknown &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: () . . () <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 42 gaag acacaa ttgg catact tcactgatag caagaatact gg gaggcaac taaaagatac 60

<210> SEQ ID NO 43 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial/Unknown &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: () . . () <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA US 6,534,300 B1 107 108

-continued SEQUENCE: 43 actgcatato citcagaatgg 20

SEQ ID NO 44 LENGTH 33 TYPE DNA ORGANISM: Artificial/Unknown FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 44 tggttctgaa gottagcc.ga gatcaatacc atg 33

SEQ ID NO 45 LENGTH 40 TYPE DNA ORGANISM: Artificial/Unknown FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 45 tagtacactc tag actacta cittcaatttgtctogataag 40

SEQ ID NO 46 LENGTH 218 TYPE DNA ORGANISM: hybrid FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: mouse/human hybrid <400 SEQUENCE: 46 citagcc.gc.ca ccatggagac agacacactc ctd citat ggg tactgct got cqgcggtggit 60 accitctgtct gtgtgaggac gatac coatg acgacgagtg ggttcCaggit to cactdgtg 120 acgaagat.ca ggtagatc.cg cqgttaatca cccaaggtoc aaggtgacca citgcttctag 18O to catctagg cqccaattag gacgg tact g c catt.cga 218

SEQ ID NO 47 LENGTH 2.05 TYPE DNA ORGANISM: hybrid FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: mouse/human hybrid <400 SEQUENCE: 47 citagcggtac catgagatta gcagtagg.cg cct tattagt atgcgcagta citcc.gc.catg 60 gtactictaat cqtcatcc.gc ggaataatca tacgc.gtcat gagggattat gtc.tc.gcaga 120 agat caggta gatcc.gcggit taatcgacgg taccttatac agagcgtctt citagt coatc 18O tagg.cgc.caa ttagct gcca titcga 2O5

SEQ ID NO 48 LENGTH: 2O7 TYPE DNA ORGANISM: hybrid US 6,534,300 B1 109 110

-continued

FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: mouse/human hybrid

<400 SEQUENCE: 48 citagcc.gc.ca ccatgggatt agcagtaggc gccittattag tatgcgcagt c gcc.ggtggit 60 accotaatcg tdatcc.gcgg aataatcata cqcgtcaact c ggattatgt citc.gcagaag 120 atcaggtaga toc goggitta atcgacgtga gcc taataca gag.cgtottc tag to catct 18O aggc.gc.ca at tagctg.cgta cattcga 2O7

SEQ ID NO 49 LENGTH: 31 TYPE DNA ORGANISM: Artificial/Unknown FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 49 ggaatticcac catggc gacc tocacggg to g 31

SEQ ID NO 50 LENGTH 19 TYPE DNA ORGANISM: Artificial/Unknown FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 50 tgaccagggit cocgtogcg 19

SEQ ID NO 51 LENGTH 39 TYPE DNA ORGANISM: Artificial/Unknown FEATURE: NAME/KEY: misc feature LOCATION: () . . () OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA

<400 SEQUENCE: 51 gaggaccagg togg acco cag gotgatccac ggcaaggat 39

SEQ ID NO 52 LENGTH 13 TYPE PRT ORGANISM: Homo sapiens

<400 SEQUENCE: 52 Glu Asp Glin Val Asp Pro Arg Lieu. Ile Asp Gly Lys Asp 1 5 10 US 6,534,300 B1 111 112 I claim: 14. A method of preparing a phosphorylated lySOSomal 1. A method of modifying a lysosomal hydrolase com hydrolase comprising contacting Said lySOSomal hydrolase prising contacting Said lySOSomal hydrolase with an isolated with an isolated N-acetylglucosamine-1-phosphodiester N-acetylglucosamine-1-phosphotransferase to produce a Cl-N-Acetylglucosamindase, wherein Said lySOSomal hydro modified lysosomal hydrolase, where in Said lase comprises a terminal mannose 6-phosphate, and Said N-acetylglucosamine-phosphotransferase comprises SEQ N-acetylglucosamine-1-phospho die Ster C.-N- ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Acetylglucosamindase comprises the amino acid Sequence 2. The method of claim 1, further comprising purifying in SEO ID NO:6. Said modified lySOSomal hydrolase after said contacting. 15. The method of claim 14, wherein said method further 3. The method of claim 1, wherein said lysosomal hydro comprises purifying the phosphorylated lySOSomal hydro lase comprises an asparagine-linked oligosaccharide with a lase. high mannose Structure. 16. The method of claim 14, wherein said lysosomal 4. The method of claim 1, where in Said hydrolase is Selected from the group consisting of N-acetylglucosamine-phosphotransferase catalyzes the C-glucosidase, C.-iduronidase, C.-galactosidase A, transfer of N-acetylglucosamine-1-phosphate from UDP-N- 15 arylsulfatase, N-acetly galactosamine-6-sulfatase, Acetylglucosamine to a mannose on the hydrolase. B-galactosidase, iduronate 2-sulfatase, ceramidase, 5. The method of claim 1, wherein said lysosomal hydro galacto cerebrosidase, B-glucoronidase, Heparan lase is a recombinant hydrolase. N-Sulfatase, N-Acetyl-O-glucosaminidase, Acetyl CoA-C.- 6. The method of claim 1, where in Said glucosaminide N-acetyl transferase, N-acetyl N-acetylglucosamine-phosphotransferase comprises amino glucosamine-6 Sulfatase, Galactose 6-sulfatase, Arylsulfa acids 1–928 of SEQID NO:1, amino acids 1–328 of SEQ ID tase A, , Arylsulfatase C, Arylsulfatase A NO:2, and amino acids 25-305 of SEQ ID NO:3. Cerebroside, Ganglioside, Acid B-galactosidase G 7. The method of claim 1, wherein said lysosomal hydro Galglioside, Acid 3-galactosidase, Hexosaminidase A, Hex lase is Selected from the group consisting of C-glucosidase, oS a minida Se B, C.-fucosidase, C.-N-Acetyl C.-iduronidase, C.-galactosidase A, arylsulfatase, 25 galactosaminidase, Glycoprotein Neuraminidase, Aspartyl N-acetlygalactosamine-6-sulfatase, B-galactosidase, idur glucosamine amidase, Acid Lipase, Acid Ceramidase, LySo onate 2-sulfatase, ceramidase, galactocerebrosidase, Somal Sphingomyelinase, Sphingomyelinase, and Glucocer B-glucoronidase, Heparan N-Sulfatase, N-Acetyl-C.- ebrosidase B-Glucosidase. glucosaminidase, Acetyl CoA-O-glucosaminide N-acetyl 17. The method of claim 14, where in Said transferase, N-acetyl-glucosamine-6 Sulfatase, Galactose N-acetylglucosamine-1-phospho die Ster C.-N- 6-sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase Acetylglucosamindase catalyzes the removal of C, Arylsulfatase A Cerebroside, Ganglioside, Acid N-acetylglucosamine from Said modified lySOSomal hydro f-galactosidase G. Galglioside, Acid f-galactosidase, lases and generates a terminal mannose 6-phosphate on Said Hexosaminidase A, Hexosaminidase B, C-fucosidase, Cl-N- hydrolase. Acetyl galactosaminidase, Glycoprotein Neuraminidase, 35 18. The method of claim 14, where in Said Aspartylglucosamine amidase, Acid Lipase, Acid N-acetylglucosamine-1-phospho die Ster C.-N- Ceramida Se, Ly So Somal Sphingomyelinase, Acetylglucosamindase comprises amino acids 50-515 of Sphingomyelinase, and Glucocerebrosidase B-Glucosidase. SEO ID NO:6. 8. The method of claim 7, wherein said lysosomal hydro 19. A lysosomal hydrolase obtained by the method of lase is C-glucosidase. 40 claim 14. 9. A phosphorylated purified lysosomal hydrolase com 20. The method of claim 16, wherein said lysosomal prising a mannose 6-phosphate, which comprises at least 6% hydrolase is C-glucosidase. bis-phosphorylated oligosaccharides. 21. A method of preparing a phosphorylated lySOSomal 10. The phosphorylated purified lysosomal hydrolase of hydrolase comprising: claim 9, which comprises up to 100% bis-phosphorylated 45 contacting Said lySOSomal hydrolase with an isolated oligosaccharides. N-acetylglucosamine-phosphotransferase to produce a 11. The phosphorylated purified lysosomal hydrolase of modified lysosomal hydrolase, where in Said claim 9, which comprises at least 5 mannose 6-phosphates. N-acetylglucosamine-phosphotransferase comprises 12. The phosphorylated purified lysosomal hydrolase of SEO ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and claim 9, which is Selected from the group consisting of 50 contacting Said modified lySOSomal hydrolase with an C-glucosidase, C.-iduronidase, C.-galactosidase A, isolated N-acetylglucosamine-1-phosphodiester Cl-N- arylsulfatase, N-acetly galactosamine-6-sulfatase, Acetylgluco Sam in da Se, where in Said B-galactosidase, iduronate 2-sulfatase, ceramidase, N-acetylglucosamine-1-phosphodie Ster C.-N- galacto cerebrosidase, B-glucoronidase, Heparan Acetylglucosamindase comprises the amino acid in N-Sulfatase, N-Acetyl-O-glucosaminidase, Acetyl CoA-C.- 55 SEO ID NO:6. glucosaminide N-acetyl transferase, N-acetyl 22. The method of claim 21, further comprising purifying glucosamine-6 Sulfatase, Galactose 6-sulfatase, Arylsulfa Said phosphorylated lysosomal hydrolase after Said contact tase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase A ing with the isolated N-acetylglucosamine-1-phosphodiester Cerebroside, Ganglioside, Acid B-galactosidase G Cl-N-Acetylglucosamindase. Galglioside, Acid B-galactosidase, Hexosaminidase A, Hex 60 23. The method of claim 21, further comprising purifying oS a minida Se B, C.-fucosidase, C.-N-Acetyl Said modified lySOSomal hydrolase prior to Said contacting galactosaminidase, Glycoprotein Neuraminidase, Aspartyl with the isolated N-acetylglucosamine-1-phosphodiester glucosamine amidase, Acid Lipase, Acid Ceramidase, LySo Cl-N-Acetylglucosamindase. Somal Sphingomyelinase, Sphingomyelinase, and Glucocer 24. A phosphorylated lysosomal hydrolase obtained by ebrosidase B-Glucosidase. 65 the method of claim 21. 13. The phosphorylated purified lysosomal hydrolase of 25. The method of claim 21, where in Said claim 12, which is C-glucosidase. N-acetylglucosamine-phosphotransferase comprises amino US 6,534,300 B1 113 114 acid 1–928 of SEQ ID NO:1, amino acids 1–328 of SEQ ID 31. The method of claim 29, wherein said lysosomal NO:2, and amino acids 25-305 of SEQ ID NO:3. hydrolase comprises an asparagine-linked oligosaccharide 26. The method of claim 21, where in Said with a high mannose Structure. N-acetylglucosamine-1-phospho die Ster C.-N- 32. The method of claim 29, wherein said lysosomal Acetylglucosamindase comprises amino acids 50-515 of 5 hydrolase is a recombinant hydrolase. SEO ID NO:6. 33. The method of claim 29, wherein said lysosomal 27. The method of claim 21, wherein said lysosomal hydrolase is Selected from the group consisting of hydrolase is Selected from the group consisting of C-glucosidase, C.-iduronidase, C.-galactosidase A, C-glucosidase, C.-iduronidase, C.-galactosidase A, arylsulfatase, N-acetly galactosamine-6-sulfatase, arylsulfatase, N-acetly galactosamine-6-sulfatase, B-galactosidase, iduronate 2-sulfatase, ceramidase, B-galactosidase, iduronate 2-sulfatase, ceramidase, galacto cerebrosidase, B-glucoronidase, Heparan galacto cerebrosidase, B-glucoronidase, Heparan N-Sulfatase, N-Acetyl-O-glucosaminidase, Acetyl CoA-C.- N-Sulfatase, N-Acetyl-O-glucosaminidase, Acetyl CoA-C.- glucosaminide N-acetyl transferase, N-acetyl glucosaminide N-acetyl transferase, N-acetyl glucosamine-6 Sulfatase, Galactose 6-sulfatase, Arylsulfa glucosamine-6 Sulfatase, Galactose 6-sulfatase, Arylsulfa 15 tase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase A tase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase A Cerebroside, Ganglioside, Acid 3-galactosidase GM, Cerebroside, Ganglioside, Acid B-galactosidase G Galglioside, Acid 3-galactosidase, Hexosaminidase A, Hex Galglioside, Acid B-galactosidase, Hexosaminidase A, Hex oS a minida Se B, C.-fucosidase, C.-N-Acetyl oS a minida Se B, C.-fucosidase, C.-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartyl galactosaminidase, Glycoprotein Neuraminidase, Aspartyl glucosamine amidase, Acid Lipase, Acid Ceramidase, LySo glucosamine amidase, Acid Lipase, Acid Ceramidase, LySo Somal Sphingomyelinase, Sphingomyelinase, and Glucocer Somal Sphingomyelinase, Sphingomyelinase, and Glucocer ebrosidase B-Glucosidase. ebrosidase B-Glucosidase. 34. The method of claim 33, wherein said lysosomal 28. The method of claim 27, wherein said lysosomal hydrolase is C-glucosidase. hydrolase is C-glucosidase. 25 35. The method of claim 29, further comprising contact 29. A method of modifying a lysosomal hydrolase com ing Said modified lysosomal hydrolase with an isolated prising contacting Said lySOSomal hydrolase with an isolated N-acetylglucosamine-1-phospho die Ster C.-N- N-acetylglucosamine-1-phosphotransferase to produce a Acetylglucosamindase, which comprises an amino acid modified lysosomal hydrolase, where in Said sequence that is at least 70% identical to SEQ ID NO:6, and N-acetylglucosamine-phosphotransferase comprises an wherein Said N-acetylglucosamine-1-phosphodiester Cl-N- amino acid sequence that is at least 70% identical to SEQ ID Acetylglucosamindase catalyzes the removal of NO:1, an amino acid sequence that is at least 70% identical N-acetylglucosamine from Said modified lySOSomal hydro to SEQID NO:2, and an amino acid sequence that is at least lases and generates a terminal mannose 6-phosphate on Said 70% identical to SEQ ID NO:3, and said hydrolase. N-acetylglucosamine-phosphotransferase catalyzes the 35 36. The method of claim 35, further comprising purifying transfer of N-acetylglucosamine-1-phosphate from UDP-N- Said lysosomal hydrolase after contacting with the isolated Acetylglucosamine to a mannose on the lySOSomal hydro N-acetylglucosamine-1-phospho die Ster C.-N- lase. Acetylglucosamindase. 30. The method of claim 29, further comprising purifying Said modified lySOSomal hydrolase after said contacting. k k k k k