Molecular Cloning and Expression of Mouse and Human cDNAs Encoding Heparan Sulfate d-Glucosaminyl 3-O-Sulfotransferase

The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPGact) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate d-glucosaminyl 3-O-sulfotransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063–27071; Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) J. Biol. Chem.271, 27072–27082). Mouse 3-OST cDNAs were isolated by proteolyzing the purified enzyme with Lys-C, sequencing the resultant peptides as well as the existing amino terminus, employing degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain full-length enzyme cDNAs from a λ Zap Express LTA cDNA library. Human 3-OST cDNAs were isolated by searching the expressed sequence tag data bank with the mouse sequence, identifying a partial-length human cDNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a λ TriplEx human brain cDNA library. The expression of wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by transient transfection of COS-7 cells and the expression of both wild-type mouse and human 3-OST by in vitrotranscription/translation demonstrate that the two cDNAs directly encode both HSact conversion and 3-OST activities. The mouse 3-OST cDNAs exhibit three different size classes because of a 5′-untranslated region of variable length, which results from the insertion of 0–1629 base pairs (bp) between residues 216 and 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3′-untranslated region ranges from 301 to 430 bp. The nucleic acid sequence of mouse and human 3-OST cDNAs are ∼85% similar, encoding novel 311- and 307-amino acid proteins of 35,876 and 35,750 daltons, respectively, that are 93% similar. The encoded enzymes are predicted to be intraluminal Golgi residents, presumably interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potentialN-glycosylation sites, which account for the apparent discrepancy between the molecular masses of the encoded enzyme (∼34 kDa) and the previously purified enzyme (∼46 kDa). The two 3-OST species also exhibit ∼50% similarity with all previously identified forms of the heparan biosynthetic enzymeN-deacetylase/N-sulfotransferase, which suggests that heparan biosynthetic enzymes share a common sulfotransferase domain. The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPGact) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate d-glucosaminyl 3-O-sulfotransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063–27071; Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) J. Biol. Chem.271, 27072–27082). Mouse 3-OST cDNAs were isolated by proteolyzing the purified enzyme with Lys-C, sequencing the resultant peptides as well as the existing amino terminus, employing degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain full-length enzyme cDNAs from a λ Zap Express LTA cDNA library. Human 3-OST cDNAs were isolated by searching the expressed sequence tag data bank with the mouse sequence, identifying a partial-length human cDNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a λ TriplEx human brain cDNA library. The expression of wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by transient transfection of COS-7 cells and the expression of both wild-type mouse and human 3-OST by in vitrotranscription/translation demonstrate that the two cDNAs directly encode both HSact conversion and 3-OST activities. The mouse 3-OST cDNAs exhibit three different size classes because of a 5′-untranslated region of variable length, which results from the insertion of 0–1629 base pairs (bp) between residues 216 and 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3′-untranslated region ranges from 301 to 430 bp. The nucleic acid sequence of mouse and human 3-OST cDNAs are ∼85% similar, encoding novel 311- and 307-amino acid proteins of 35,876 and 35,750 daltons, respectively, that are 93% similar. The encoded enzymes are predicted to be intraluminal Golgi residents, presumably interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potentialN-glycosylation sites, which account for the apparent discrepancy between the molecular masses of the encoded enzyme (∼34 kDa) and the previously purified enzyme (∼46 kDa). The two 3-OST species also exhibit ∼50% similarity with all previously identified forms of the heparan biosynthetic enzymeN-deacetylase/N-sulfotransferase, which suggests that heparan biosynthetic enzymes share a common sulfotransferase domain. The serine proteases of the intrinsic blood coagulation cascade are slowly neutralized by antithrombin (AT) 1The abbreviations used are: AT, antithrombin; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; HSact, anticoagulant heparan sulfate; HSPGact, anticoagulant heparan sulfate proteoglycan(s); HSinact, nonanticoagulant heparan sulfate; HSPGinact, nonanticoagulant heparan sulfate proteoglycan; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; GAG(s), glycosaminoglycan(s); PBS, phosphate-buffered saline; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; 3-OST, heparan sulfate glucosaminyl 3-O-sulfotransferase; r3-OST, recombinantly expressed 3-OST enzyme; NST,N-deacetylase/N-sulfotransferase; IdceA, iduronic acid; AMN, anhydromannitol; GlcA, glucuronic acid; HPLC, high pressure liquid chromatography; PCR, polymerase chain reaction; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid; RPIP-HPLC, reverse phase ion pairing HPLC; bp, base pair(s); kb, kilobase pair(s). (reviewed in Ref. 1Rosenberg R.D. Annu. Rev. Med. 1978; 29: 367-378Crossref PubMed Scopus (52) Google Scholar). This inhibition is secondary to the generation of 1:1 enzyme·AT complexes whose formation is dramatically enhanced by the mast cell product, heparin (2Rosenberg R.D. Damus P.S. J. Biol. Chem. 1973; 248: 6490-6505Abstract Full Text PDF PubMed Google Scholar). Damus et al. (3Damus P.S. Hicks M. Rosenberg R.D. Nature. 1973; 246: 355-357Crossref PubMed Scopus (422) Google Scholar) hypothesized that endothelial cell surface heparan sulfate proteoglycans (HSPGs) function in a similar fashion to accelerate coagulation enzyme inactivation by AT and therefore are responsible for the nonthrombogenic properties of blood vessels. We initially demonstrated that perfusion of the hind limbs of normal rodents and rodents deficient in mast cells with purified thrombin and AT leads to a greatly elevated rate of thrombin·AT complex formation and that the enzyme heparitinase as well as the natural heparin antagonist platelet factor 4 suppress the above acceleration (4Marcum J.A. McKenney J.B. Galli S.J. Jackman R.W. Rosenberg R.D. Am. J. Physiol. 1986; 250: H879-H888PubMed Google Scholar, 5Marcum J.A. McKenney J.B. Rosenberg R.D. J. Clin. Invest. 1984; 74: 341-350Crossref PubMed Scopus (234) Google Scholar). We subsequently showed that cultured cloned bovine macrovascular and rodent microvascular endothelial cells synthesize both anticoagulant HSPG (HSPGact) and nonanticoagulant HSPG (HSPGinact) (6Marcum J.A. Atha D.H. Fritze L.M.S. Nawroth P. Stern D. Rosenberg R.D. J. Biol. Chem. 1986; 261: 7507-7517Abstract Full Text PDF PubMed Google Scholar, 7Marcum J.A. Rosenberg R.D. Biochem. Biophys. Res. Commun. 1985; 126: 365-372Crossref PubMed Scopus (145) Google Scholar, 8Kojima T. Leone C.W. Marchildon G.A. Marcum J.A. Rosenberg R.D. J. Biol. Chem. 1992; 267: 4859-4869Abstract Full Text PDF PubMed Google Scholar). HSPGact bear glycosaminoglycan (GAG) chains that bind tightly to AT and accelerate thrombin·AT complex generation (6Marcum J.A. Atha D.H. Fritze L.M.S. Nawroth P. Stern D. Rosenberg R.D. J. Biol. Chem. 1986; 261: 7507-7517Abstract Full Text PDF PubMed Google Scholar, 7Marcum J.A. Rosenberg R.D. Biochem. Biophys. Res. Commun. 1985; 126: 365-372Crossref PubMed Scopus (145) Google Scholar, 8Kojima T. Leone C.W. Marchildon G.A. Marcum J.A. Rosenberg R.D. J. Biol. Chem. 1992; 267: 4859-4869Abstract Full Text PDF PubMed Google Scholar). The biosynthesis of HSPGact requires generation of a core protein; assembly of a linkage region of four neutral sugars on specific serine attachment sites of the core protein; elongation of a GAG backbone composed of alternating N-acetylglucosamine and glucuronic acid residues; and modification of this homogenous copolymer by partial N-deacetylation with coupledN-sulfation of glucosamine residues, partial epimerization of glucuronic acid to iduronic acid residues, partial 2-O-sulfation of uronic acid residues, and partial 6-O-sulfation and partial 3-O-sulfation of glucosamine residues (reviewed in Ref. 9Lindahl U. Kjellén L. Wight T.N. Mecham R. The Biology of the Extracellular Matrix Proteoglycans. Academic Press, Inc., New York1987: 59-104Google Scholar). This multienzyme pathway generates HSPGact with regions of defined structure that contain the primary AT binding domain sequence found in anticoagulant heparin: uronic acid → glucosamine (N-acetyl/N-sulfate) 6-O-sulfate → glucuronic acid → glucosamine N-sulfate 3-O-sulfate (6-O-sulfate) → iduronic acid 2-O-sulfate → glucosamine N-sulfate 6-O-sulfate (10Atha D.H. Stevens A.W. Rimon A. Rosenberg R.D. Biochemistry. 1984; 23: 5801-5812Crossref PubMed Scopus (57) Google Scholar, 11Atha D.H. Lormeau J.C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1985; 24: 6723-6729Crossref PubMed Scopus (189) Google Scholar, 12Atha D.H. Lormeau J.C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1987; 26: 6454-6461Crossref PubMed Scopus (115) Google Scholar, 13Choay J. Petitou M. Lormeau J.C. Sinaÿ P. Casu B. Gatti G. Biochem. Biophys. Res. Commun. 1983; 116: 492-499Crossref PubMed Scopus (596) Google Scholar, 14Lindahl U. Bäckström G. Thunberg L. Leder I.G. Proc. Nat. Acad. Sci. U. S. A. 1980; 77: 6551-6555Crossref PubMed Scopus (418) Google Scholar, 15Lindahl U. Bäckström G. Thunberg L. J. Biol. Chem. 1983; 258: 9826-9830Abstract Full Text PDF PubMed Google Scholar, 16Rosenberg R.D. Lam L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1218-1222Crossref PubMed Scopus (232) Google Scholar, 17Rosenberg R.D. Armand G. Lam L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3065-3069Crossref PubMed Scopus (108) Google Scholar). These reactions also produce HSPGinact with regions of varying monosaccharide sequence that lack the primary AT-binding domain. The structure-function relationships of the AT binding domain have been elucidated with heparin/heparan sulfate oligosaccharides in association with fast reaction kinetics and equilibrium binding assays. The 6-O-sulfate group on residue 2 and the 3-O-sulfate group on residue 4 function in a thermodynamically linked fashion to supply half of the binding energy for interaction with AT and trigger a conformational event that accelerates neutralization of specific coagulation proteases (11Atha D.H. Lormeau J.C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1985; 24: 6723-6729Crossref PubMed Scopus (189) Google Scholar, 12Atha D.H. Lormeau J.C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1987; 26: 6454-6461Crossref PubMed Scopus (115) Google Scholar). The amino and ester sulfate groups at residues 5 and 6 as well as carboxyl groups at other sites provide the other half of the binding energy for interaction with protease inhibitor (10Atha D.H. Stevens A.W. Rimon A. Rosenberg R.D. Biochemistry. 1984; 23: 5801-5812Crossref PubMed Scopus (57) Google Scholar, 11Atha D.H. Lormeau J.C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1985; 24: 6723-6729Crossref PubMed Scopus (189) Google Scholar). Furthermore, monosaccharide sequences outside the primary AT binding domain are essential in facilitating inhibition of coagulation proteases other than factor Xa (18Oosta G.M. Gardner W.T. Beeler D.L. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 829-833Crossref PubMed Scopus (148) Google Scholar, 19Stone A.L. Beeler D.L. Oosta G.M. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7190-7194Crossref PubMed Scopus (50) Google Scholar). During the past 8 years, several biosynthetic enzymes that generate HSPGact and HSPGinact have been purified. These proteins include the N-acetylglucosamine/glucuronic acid copolymerase (20Lind T. Lindahl U. Lidholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar), theN-deacetylase/N-sulfotransferase (NST) (21Brandan E. Hirschberg C.B. J. Biol. Chem. 1988; 263: 2417-2422Abstract Full Text PDF PubMed Google Scholar, 22Pettersson I. Kusche M. Unger E. Wlad H. Nylund L. Lindahl U. Kjellén L. J. Biol. Chem. 1991; 266: 8044-8049Abstract Full Text PDF PubMed Google Scholar), the glucuronic acid/iduronic acid epimerase (23Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J.-P. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar), the iduronic acid/glucuronic acid 2-O-sulfotransferase (24Kobayashi M. Habuchi H. Habuchi O. Saito M. Kimata K. J. Biol. Chem. 1996; 271: 7645-7653Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), the glucosamine 6-O-sulfotransferase (25Habuchi H. Habuchi O. Kimata K. J. Biol. Chem. 1995; 270: 4172-4179Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and the glucosamine 3-O-sulfotransferase (3-OST) (26Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, the only enzymes that have also been molecularly cloned are two structurally and functionally distinct isoforms of theN-deacetylase/N-sulfotransferase (NST-1 from liver and NST-2 from mastocytoma) (27Orellana A. Hirschberg C.B. Wei Z. Swiedler S.J. Ishihara M. J. Biol. Chem. 1994; 269: 2270-2276Abstract Full Text PDF PubMed Google Scholar, 28Hashimoto Y. Orellana A. Gil G. Hirschberg C.B. J. Biol. Chem. 1992; 267: 15744-15750Abstract Full Text PDF PubMed Google Scholar, 29Eriksson I. Sandbäck D. Ek B. Lindahl U. Kjellén L. J. Biol. Chem. 1994; 269: 10438-10443Abstract Full Text PDF PubMed Google Scholar, 30Ishihara M. Guo Y. Wei Z. Yang Z. Swiedler S.J. Orellana A. Hirschberg C.B. J. Biol. Chem. 1993; 268: 20091-20095Abstract Full Text PDF PubMed Google Scholar, 31Cheung W.-F. Eriksson I. Kusche-Gullberg M. Lindahl U. Kjellén L. Biochemistry. 1996; 35: 5250-5256Crossref PubMed Scopus (49) Google Scholar). The heparan biosynthetic enzymes must function in a coordinated manner to produce the AT binding domain, because the abundance of this sequence is much greater than predicted from a random assembly of constituents (32Casu B. Adv. Carbohydr. Chem. Biochem. 1985; 43: 51-134Crossref PubMed Scopus (430) Google Scholar). The postulated regulatory mechanism must direct the biosynthetic enzymes to carry out the appropriate sequence of epimerization/sulfation reactions to generate the AT binding domain (33Shworak N.W. Shirakawa M. Colliec-Jouault S. Liu J. Mulligan R.C. Birinyi L.K. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24941-24952Abstract Full Text PDF PubMed Google Scholar, 34Colliec-Jouault S. Shworak N.W. Liu J. de Agostini A.I. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24953-24958Abstract Full Text PDF PubMed Google Scholar). We have previously described a soluble cell-free system to investigate HSact generation and developed assays for defining critical enzymatic components (35Shworak N.W. Fritze L.M.S. Liu J. Butler L.D. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27063-27071Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The investigations employing the above approach define a limiting HSact conversion activity that acts upon an excess precursor population to regulate cellular HSPGact biosynthesis (35Shworak N.W. Fritze L.M.S. Liu J. Butler L.D. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27063-27071Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The major component of the limiting HSact conversion activity proved to be the long sought 3-OST, as documented by purification and characterization of this protein (26Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The investigations utilizing the above technique also showed that HSact precursor (35% of the HSinact pool) is 3-O-sulfated to generate HSPGact and that HSinact precursor (65%) is 3-O-sulfated to produce HSPGinact. However, only a small fraction of either substrate is modified, with the remaining precursor population exiting the Golgi apparatus to appear on the cell surface. Thus, 3-OST constitutes a rate-limiting enzymatic activity that defines the level of cellular HSPGact generation. In contrast, the level of 3-OST activity does not appear to limit the mast cell formation of anticoagulant heparin, which contains a high proportion of molecules with the AT binding site (∼30%) (36Lam L.H. Silbert J.E. Rosenberg R.D. Biochem. Biophys. Res. Commun. 1976; 69: 570-577Crossref PubMed Scopus (370) Google Scholar); structural and biochemical analyses indicate that the precursor of anticoagulant heparin is present in minimal amounts (37Linhardt R.J. Wang H. Loganathan D. Bae J. J. Biol. Chem. 1992; 267: 2380-2387Abstract Full Text PDF PubMed Google Scholar, 38Kusche M. Torri G. Casu B. Lindahl U. J. Biol. Chem. 1990; 265: 7292-7300Abstract Full Text PDF PubMed Google Scholar). Despite this biosynthetic difference, for both heparin and heparan, 3-O-sulfation does not guarantee the formation of anticoagulant GAG (33Shworak N.W. Shirakawa M. Colliec-Jouault S. Liu J. Mulligan R.C. Birinyi L.K. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24941-24952Abstract Full Text PDF PubMed Google Scholar, 34Colliec-Jouault S. Shworak N.W. Liu J. de Agostini A.I. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24953-24958Abstract Full Text PDF PubMed Google Scholar, 39Montgomery R. Lidholt K. Flay N. Liang J. Vertel B. Lindahl U. Esko J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11327-11331Crossref PubMed Scopus (28) Google Scholar, 40Razi N. Lindahl U. J. Biol. Chem. 1995; 270: 11267-11275Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This phenomenon may be secondary to alterations in the relative concentrations of two functional forms of 3-OST that differentially act upon HSact precursorversus HSinact precursor or changes in the relative levels of HSact precursor versus the HSinact precursor. The two functional forms of 3-OST may be due to two discrete gene products, posttranslational modification of a single gene product, or the presence of a regulatory factor that directs the enzyme to modify one or the other precursor. The relative levels of HSact precursor versusHSinact precursor are presumably controlled by earlier biosynthetic enzymes. In the current paper, we molecularly clone as well as express murine and human 3-OST, and we show that the expressed enzyme is able to 3-O-sulfate both HSactprecursor and HSinact precursor. Furthermore, the deduced structure of 3-OST, when compared with NST, defines a heparan sulfate sulfotransferase domain and also suggests a novel mechanism for limiting the action of the enzyme. We have previously described the clonal L cell line LTA (35Shworak N.W. Fritze L.M.S. Liu J. Butler L.D. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27063-27071Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,41de Agostini A.L. Lau H.K. Leone C. Youssoufian H. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9784-9788Crossref PubMed Scopus (22) Google Scholar), the generation of clone 33, an LTA transfectant that overexpresses the ryudocan12CA5 cDNA (33Shworak N.W. Shirakawa M. Colliec-Jouault S. Liu J. Mulligan R.C. Birinyi L.K. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24941-24952Abstract Full Text PDF PubMed Google Scholar), a rapidly growing revertant of clone 33, L-33+ (26Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and RFPEC, an immortalized line derived from rat fat pad endothelial cells (8Kojima T. Leone C.W. Marchildon G.A. Marcum J.A. Rosenberg R.D. J. Biol. Chem. 1992; 267: 4859-4869Abstract Full Text PDF PubMed Google Scholar). Primary mouse neonatal endothelial cells from the cardiac microvasculature of day 3–5 neonates (CME cells) were a generous gift from Dr. Jay Edelberg (MIT/Beth Israel Hospital), whereas COS-7 cells were obtained from the ATCC. Primary human umbilical vein endothelial cells (HUVEC) were maintained according to the supplier's (Clonetics Corp., San Diego, CA) protocol. Unless otherwise stated, all cell lines were maintained in logarithmic growth by subculturing biweekly in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C under 5% CO2 humidified atmosphere, as described previously (42Shworak N.W. Shirakawa M. Mulligan R.C. Rosenberg R.D. J. Biol. Chem. 1994; 269: 21204-21214Abstract Full Text PDF PubMed Google Scholar). Exponentially growing cultures were generated by inoculating 54,000 cells/cm2 and incubating for 2 days, whereas postconfluent cultures were produced by inoculating 250,000 cells/cm2 and allowing growth for 10 days with medium exchanges on days 4, 7, 8, and 9. The purification of mouse 3-OST froml-33+ has been previously described (26Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and the final step 4 product was concentrated by reverse phase chromatography on an HP 1090 M system (Hewlett Packard) equipped with a C4 reverse phase HPLC column (250 × 2.1-mm, 300-Å pore size, 5-μm particle size) (Vydac, catalog number 214TP52) equilibrated in 1.6% acetonitrile (v/v), 0.1% trifluoroacetic acid (v/v). After application of the sample, the reverse phase matrix was washed with 60% acetonitrile, 0.1% trifluoroacetic acid, and bound species were eluted with 78.4% acetonitrile, 0.1% trifluoroacetic acid. Samples of 1.5 or 3 μg, from two independent purifications, were digested with 0.15 or 0.3 μg, respectively, of endopeptidase Lys-C (Waco) in a reaction volume of 100 μl containing 1% RTX100 (Calbiochem), 10% acetonitrile, and 100 mm Tris-HCl, pH 8.0, at 37 °C for ∼16 h (43Fernandez J. DeMott M. Atherton D. Mische S.M. Anal. Biochem. 1992; 201: 255-264Crossref PubMed Scopus (247) Google Scholar). Digestion products were chromatographed on a HP 1090 M system (Hewlett Packard) equipped with the above described C4 reverse phase HPLC column equilibrated in 98% trifluoroacetic acid acetonitrile (v/v), trifluoroacetic acid After application of products, the reverse phase matrix was washed with 98% and bound species were eluted with of to to and to 98% G. 1990; PubMed Scopus (28) Google Scholar). The was for at and and were and with a In the sequence of of concentrated 3-OST was directly was isolated from postconfluent cultures of LTA cells of × cells) by a modification of the of al. J. R. R. 1980; PubMed Scopus Google Scholar). were washed with PBS, cells were by and for 2 and cell were washed by in by × for 4 were by for in of mm pH mm 5 mm 1% 5 mm complexes (Life were on for 10 and for were by at × for 10 the was with an volume of mm pH mm mm containing μg/ml and the was at °C for 2 Samples were an volume of the phase was with of was by at for 10 and was in of 10 mm pH mm was isolated from of by two 100 of (Life Technologies, catalog number according to the that binding and 0.1% and was for The final was 1.5 of the phase was to 100 mm and mm an volume of was the was at × for and the was in μl of primers were obtained from cDNA was generated in a volume from 5 of LTA with a reverse according to the 1996; PubMed Scopus Google Scholar, K. Res. 1991; PubMed Scopus Google Scholar) reactions μl of cDNA, of μl of a of and × distinct of were to obtain of with primers and reactions were to °C for to of °C for and °C for with a °C by of °C for and °C for with a °C and °C for and of °C for °C for 10 and to °C for with primers and or primers and reactions were to °C for 4 to of °C for and °C for 2 with °C and by of °C for °C for and to °C products were purified as the from a molecular membrane catalog number and of was with polymerase and catalog number according to the the product of bp, which was by with and and isolated by probe was generated with a random catalog number by the random primers with 5 of the an λ Zap Express cDNA catalog number was generated from 5 of LTA that been with 1.5 × primary were by the × were to a and with the above described probe specific for were at °C in × and were washed with 2 × 1% for at were and in by with by of E. The for data bank of the expressed sequence tag cDNA G. C. M. 1996; PubMed Scopus Google Scholar) was with the deduced mouse 3-OST amino acid sequence to three partial-length