Lysosomal and Cytosolic Sialic Acid 9-O-Acetylesterase Activities Can Be Encoded by One Gene via Differential Usage of a Signal Peptide-encoding Exon at the N Terminus

9-O-Acetylation is one of the most common modifications of sialic acids, and it can affect several sialic acid-mediated recognition phenomena. We previously reported a cDNA encoding a lysosomal sialic acid-specific 9-O-acetylesterase, which traverses the endoplasmic reticulum-Golgi pathway and localizes primarily to lysosomes and endosomes. In this study, we report a variant cDNA derived from the same gene that contains a different 5′ region. This cDNA has a putative open reading frame lacking a signal peptide-encoding sequence and is thus a candidate for the previously described cytosolic sialic acid 9-O-acetylesterase activity. Epitope-tagged constructs confirm that the new sequence causes the protein product to be targeted to the cytosol and has esterase activity. Using reverse transcription-polymerase chain reaction to distinguish the two forms of message, we show that although the lysosomal sialic acid-specific 9-O-acetylesterase message has a widespread pattern of expression in adult mouse tissues, this cytosolic sialic acid 9-O-acetylesterase form has a rather restricted distribution, with the strongest expression in the liver, ovary, and brain. Using a polyclonal antibody directed against the 69-amino acid region common to both proteins, we confirmed that the expression of glycosylated and nonglycosylated polypeptides occurred in appropriate subcellular fractions of normal mouse tissues. Rodent liver polypeptides reacting to the antibody also co-purify with previously described lysosomal sialic acid esterase activity and at least a portion of the cytosolic activity. Thus, two sialic acid 9-O-acetylesterases found in very different subcellular compartments can be encoded by a single gene by differential usage of a signal peptide-encoding exon at the N terminus. The 5′-rapid amplification of cDNA ends results and the differences in tissue-specific expression suggest that expression of these two products may be differentially regulated by independent promoters. 9-O-Acetylation is one of the most common modifications of sialic acids, and it can affect several sialic acid-mediated recognition phenomena. We previously reported a cDNA encoding a lysosomal sialic acid-specific 9-O-acetylesterase, which traverses the endoplasmic reticulum-Golgi pathway and localizes primarily to lysosomes and endosomes. In this study, we report a variant cDNA derived from the same gene that contains a different 5′ region. This cDNA has a putative open reading frame lacking a signal peptide-encoding sequence and is thus a candidate for the previously described cytosolic sialic acid 9-O-acetylesterase activity. Epitope-tagged constructs confirm that the new sequence causes the protein product to be targeted to the cytosol and has esterase activity. Using reverse transcription-polymerase chain reaction to distinguish the two forms of message, we show that although the lysosomal sialic acid-specific 9-O-acetylesterase message has a widespread pattern of expression in adult mouse tissues, this cytosolic sialic acid 9-O-acetylesterase form has a rather restricted distribution, with the strongest expression in the liver, ovary, and brain. Using a polyclonal antibody directed against the 69-amino acid region common to both proteins, we confirmed that the expression of glycosylated and nonglycosylated polypeptides occurred in appropriate subcellular fractions of normal mouse tissues. Rodent liver polypeptides reacting to the antibody also co-purify with previously described lysosomal sialic acid esterase activity and at least a portion of the cytosolic activity. Thus, two sialic acid 9-O-acetylesterases found in very different subcellular compartments can be encoded by a single gene by differential usage of a signal peptide-encoding exon at the N terminus. The 5′-rapid amplification of cDNA ends results and the differences in tissue-specific expression suggest that expression of these two products may be differentially regulated by independent promoters. sialic acid cytosolic sialic acid 9-O-acetylesterase lysosomal sialic acid-specific 9-O-acetylesterase reverse transcription polymerase chain reaction Concanavalin A base pair(s) polyacrylamide gel electrophoresis peptide N-glycosidase F Sialic acids (Sias)1 are a diverse family of acidic 9-carbon sugars typically found at the nonreducing end of sugar chains of animals throughout the deuterostome lineage (1Schauer R. Sialic Acids: Chemistry, Metabolism and Function, Cell Biology Monographs. 10. Springer-Verlag, New York1982Crossref Google Scholar, 2Varki A. Glycobiology. 1992; 2: 25-40Crossref PubMed Scopus (490) Google Scholar). Sias are now recognized not only as molecules responsible for negative charge and hydrophilicity of the cell surface but also as specific ligands playing important roles in intercellular and/or intermolecular recognition phenomena (3Varki A. FASEB J. 1997; 11: 248-255Crossref PubMed Scopus (493) Google Scholar). There have been several vertebrate receptors reported for Sia-containing determinants, including the selectin family (4Kansas G.S. Blood. 1996; 88: 3259-3287Crossref PubMed Google Scholar, 5McEver R.P. Cummings R.D. J. Clin. Invest. 1997; 100: 485-492Crossref PubMed Google Scholar, 6Varki A. J. Clin. Invest. 1997; 99: 158-162Crossref PubMed Scopus (261) Google Scholar) and the Siglec family (7Crocker P.R. Clark E.A. Filbin M. Gordon S. Jones Y. Kehrl J.H. Kelm S. Le Douarin N. Powell L. Roder J. Schnaar R.L. Sgroi D.C. Stamenkovic K. Schauer R. Schachner M. Van den Berg T.K. Van der Merwe P.A. Watt S.M. Varki A. Glycobiology. 1998; 8: vCrossref PubMed Google Scholar, 8Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 14243-14246Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 9Kelm S. Schauer R. Int. Rev. Cytol. 1997; 175: 137-240Crossref PubMed Google Scholar, 10Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar). Sias are also known to be targets for a variety of microbial proteins involved in host cell recognition, e.g. the influenza virus hemagglutinin (11Herrler G. Rott R. Klenk H.D. Muller H.P. Shukla A.K. Schauer R. EMBO J. 1985; 4: 1503-1506Crossref PubMed Scopus (198) Google Scholar) and one of the adhesins of Helicobacter pylori (12Miller-Podraza H. Abul Milh M. Teneberg S. Karlsson K.A. Infect. Immun. 1997; 65: 2480-2482Crossref PubMed Google Scholar, 13Miller-Podraza H. Bergstrom J. Milh M.A. Karlsson K.A. Glycoconj. J. 1997; 14: 467-471Crossref PubMed Scopus (33) Google Scholar, 14Johansson L. Karlsson K.A. Glycoconj. J. 1998; 15: 713-721Crossref PubMed Scopus (17) Google Scholar). The Sia molecule can be modified by the addition of O-acetyl esters on the hydroxyl-groups of the 4, 7, 8, and 9 positions (2Varki A. Glycobiology. 1992; 2: 25-40Crossref PubMed Scopus (490) Google Scholar). This O-acetylation of Sia is a common modification found in mammalian cell surface sialoglycoconjugates and can also be present on free Sias in the cytosol (15Schauer R. Hoppe. Seylers. Z. Physiol. Chem. 1970; 351: 749-758Crossref PubMed Scopus (34) Google Scholar, 16Schauer R. Wember M. Hoppe-Seyler's Z. Physiol. Chem. 1971; 352: 1282-1290Crossref PubMed Scopus (33) Google Scholar). One example of a well characterized O-acetylated sialoglycoconjugate is 9(7)-O-acetylated-GD3 and structurally related disialogangliosides, which show developmentally regulated expression and gradient patterns for its staining with monospecific antibodies in the developing brain (17Schlosshauer B. Blum A.S. Mendez Otero R. Barnstable C.J. Constantine-Paton M. J. Neurosci. 1988; 8: 580-592Crossref PubMed Google Scholar, 18Sparrow J.R. Barnstable C.J. J. Neurosci. 1988; 21: 398-409Google Scholar). Specific antibodies showed that the expression of 9-O-acetylated GD3 appears to be independently regulated from that of the parental molecule, GD3 (18Sparrow J.R. Barnstable C.J. J. Neurosci. 1988; 21: 398-409Google Scholar), and occurs on the migrating neural cells (19Blum A.S. Barnstable C.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8716-8720Crossref PubMed Scopus (99) Google Scholar). Despite the widespread and regulated occurrence of this modification, the biological significance of O-acetylation is largely unknown. It has been shown that O-acetylation of Sia can mask the Sia-containing determinant from recognizing its counter receptor lectin Siglec-2 (CD22) (20Sjoberg E.R. Powell L.D. Klein A. Varki A. J. Cell Biol. 1994; 126: 549-562Crossref PubMed Scopus (171) Google Scholar). Direct or indirect evidence indicates that binding of other members of the Siglec family to their sialylated ligands can also be blocked by O-acetylation (21Collins B.E. Yang L.J.S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Collins B.E. Kiso M. Hasegawa A. Tropak M.B. Roder J.C. Crocker P.R. Schnaar R.L. J. Biol. Chem. 1997; 272: 16889-16895Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 23Kelm S. Brossmer R. Isecke R. Gross H.J. Strenge K. Schauer R. Eur. J. Biochem. 1998; 255: 663-672Crossref PubMed Scopus (144) Google Scholar, 24Strenge K. Schauer R. Bovin N. Hasegawa A. Ishida H. Kiso M. Kelm S. Eur. J. Biochem. 1998; 258: 677-685Crossref PubMed Scopus (57) Google Scholar, 25Shi W.X. Chammas R. Varki N.M. Powell L. Varki A. J. Biol. Chem. 1996; 271: 31526-31532Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In contrast, influenza virus C hemagglutinin specifically requires 9-O-acetylated Sia for binding to host cells (11Herrler G. Rott R. Klenk H.D. Muller H.P. Shukla A.K. Schauer R. EMBO J. 1985; 4: 1503-1506Crossref PubMed Scopus (198) Google Scholar,26Muchmore E. Varki A. Science. 1987; 236: 1293-1295Crossref PubMed Scopus (72) Google Scholar). These examples indicate that O-acetylation may be a key modification regulating Sia-dependent recognition events. In fact, cleavage of 9-O-acetyl groups from Sia molecules by transgenic expression of influenza C virus hemagglutinin 9-O-acetylesterase caused abnormalities in murine development (27Varki A. Hooshmand F. Diaz S. Varki N.M. Hedrick S.M. Cell. 1991; 65: 65-74Abstract Full Text PDF PubMed Scopus (120) Google Scholar). The biosynthesis of O-acetylated sialoglycoconjugates is catalyzed by Sia-specific O-acetyltransferase(s) that use acetyl-CoA as an acetyl donor (28Diaz S. Higa H.H. Hayes B.K. Varki A. J. Biol. Chem. 1989; 264: 19416-19426Abstract Full Text PDF PubMed Google Scholar). This O-acetylation reaction appears to take place in the late Golgi apparatus, after the action of sialyltransferases (29Chammas R. McCaffery J.M. Klein A. Ito Y. Saucan L. Palade G. Farquhar M.G. Varki A. Mol. Biol. Cell. 1996; 7: 1691-1707Crossref PubMed Scopus (21) Google Scholar). Various lines of evidence indicate the existence of a family of 9(7)-O-acetyltransferase(s) that are linkage- and molecule -specific in their action, likely explaining the tissue- and stage-specific expression of this modification (30Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 15130-15138Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). These labile Golgi enzymes have so far proven refractory to purification or cloning. This modification can be removed by Sia-specific 9-O-acetylesterases. We and others have previously described two distinct forms of mammalian Sia O-acetylesterases, one in the cytosolic fraction and another in the lysosomal/endosomal compartment (31Higa H.H. Butor C. Diaz S. Varki A. J. Biol. Chem. 1989; 264: 19427-19434Abstract Full Text PDF PubMed Google Scholar, 32Schauer R. Reuter G. Stoll S. Shukla A.K. J. Biochem. (Tokyo). 1989; 106: 143-150Crossref PubMed Scopus (19) Google Scholar, 33Butor C. Higa H.H. Varki A. J. Biol. Chem. 1993; 268: 10207-10213Abstract Full Text PDF PubMed Google Scholar, 34Butor C. Griffiths G. Aronson Jr., N.N. Varki A. J. Cell Sci. 1995; 108: 2213-2219Crossref PubMed Google Scholar). This localization of the lysosomal Sia 9-O-acetylesterase (Lse) is somewhat unusual, considering its neutral pH optimum (34Butor C. Griffiths G. Aronson Jr., N.N. Varki A. J. Cell Sci. 1995; 108: 2213-2219Crossref PubMed Google Scholar). Regardless, this enzyme is likely to be the one participating in the terminal lysosomal degradation of 9-O-acetylated sialoglycoconjugates. However, it would not have access to free 9-O-acetylated Sias that have been reported in the cytosolic fraction. These presumably result from the action of lysosomal sialidases on 9-O-acetylated Sias, followed by export of such molecules to the cytosol by the action of the lysosomal Sia exporter (15Schauer R. Hoppe. Seylers. Z. Physiol. Chem. 1970; 351: 749-758Crossref PubMed Scopus (34) Google Scholar, 16Schauer R. Wember M. Hoppe-Seyler's Z. Physiol. Chem. 1971; 352: 1282-1290Crossref PubMed Scopus (33) Google Scholar). Such 9-O-acetylated Sias entering the cytosol are known to be poor substrates for reactivation by CMP-Sia synthase (35Higa H.H. Paulson J.C. J. Biol. Chem. 1985; 260: 8838-8849Abstract Full Text PDF PubMed Google Scholar), and CMP-9-O-acetylated Sias in turn are poor substrates for some sialyltransferases (35Higa H.H. Paulson J.C. J. Biol. Chem. 1985; 260: 8838-8849Abstract Full Text PDF PubMed Google Scholar). Thus, we have suggested that the function of the cytosolic Sia 9-O-acetylesterase (Cse) activity is to salvage any 9-O-acetylated molecules that escape the initial action of the Lse enzyme. The Cse and Lse enzyme activities are both found in the ultracentrifugate supernatant of freeze-thawed rat liver (the soluble Lse enzyme is presumably released by the breakage of lysosomal membranes during freeze-thaw) or in detergent extracts. The two activities can be biochemically separated by ConA-Sepharose chromatography (the Lse binds and the Cse runs through the column) and/or by DEAE-cellulose chromatography (the Cse binds and the Lse runs through) (36Higa H.H. Manzi A. Varki A. J. Biol. Chem. 1989; 264: 19435-19442Abstract Full Text PDF PubMed Google Scholar). Furthermore, the reported properties of the Cse are somewhat different from that of the Lse (32Schauer R. Reuter G. Stoll S. Shukla A.K. J. Biochem. (Tokyo). 1989; 106: 143-150Crossref PubMed Scopus (19) Google Scholar, 37Schauer R. Methods Enzymol. 1988; 138: 611-626Crossref Scopus (54) Google Scholar). Finally, some antibodies directed against the Lse did not seem to precipitate the Cse activity (33Butor C. Higa H.H. Varki A. J. Biol. Chem. 1993; 268: 10207-10213Abstract Full Text PDF PubMed Google Scholar). Based on all of these criteria, it has always been assumed that the Lse and Cse enzymes are products of separate genes. We earlier purified the Lse to homogeneity and obtained N-terminal amino acid sequences of the two subunits (33Butor C. Higa H.H. Varki A. J. Biol. Chem. 1993; 268: 10207-10213Abstract Full Text PDF PubMed Google Scholar). These sequences were subsequently used to identify the Lse cDNA, which was initially isolated serendipitously during differential display analysis of different stages of murine hematopoietic development (38Guimaraes M.J. Bazan J.F. Castagnola J. Diaz S. Copeland N.G. Gilbert D.J. Jenkins N.A. Varki A. Zlotnik A. J. Biol. Chem. 1996; 271: 13697-13705Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The cDNA encoding the Lse was also independently cloned by means of differential display comparisons between different stages of the murine B lymphoid cell lineage (39Stoddart A. Zhang Y. Paige C.J. Nucleic Acids Res. 1996; 24: 4003-4008Crossref PubMed Scopus (21) Google Scholar). During the characterization of the latter system, several cDNAs were noted to have 5′ sequences that were different from the originally isolated Lse form. Here we report the characterization of one of these cDNAs and show that it can encode a cytosolic counterpart of the Lse. All reagents used for this study were of appropriate grade for either biochemistry or molecular biology and unless otherwise stated were obtained from Sigma. Unless otherwise stated, typical experiments followed the Current Protocols in Molecular Biology (40Authors Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New-York1993Google Scholar), Current Protocols in Protein Science (41Coligan J.E. Current Protocols in Protein Science. John Wiley & Sons, Inc., Brooklyn, NY1996Google Scholar), or Molecular Cloning (42Sambrook J. Maniatis T. Fritsch E.F. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 7A3-C is a cDNA clone previously isolated from the 70Z/3 pre B-cell line cDNA library by cross-hybridization to the Lse (39Stoddart A. Zhang Y. Paige C.J. Nucleic Acids Res. 1996; 24: 4003-4008Crossref PubMed Scopus (21) Google Scholar). We subcloned the Lse into the expression vector pcDNAI/Amp (Invitrogen) after PCR-derived mutagenesis to add a FLAG epitope in its C terminus (Lse-FLAG) as reported previously (38Guimaraes M.J. Bazan J.F. Castagnola J. Diaz S. Copeland N.G. Gilbert D.J. Jenkins N.A. Varki A. Zlotnik A. J. Biol. Chem. 1996; 271: 13697-13705Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We then exchanged the 5′ region of Lse from the 7A3-C using the Bal I site within the coding sequence of both forms and Hin dIII site in the multiple cloning site of pcDNAI/Amp (giving Cse-C-FLAG). Plasmid DNA was prepared using a Qiagen maxi-column (Qiagen) and transiently transfected into COS cells using LipofectAMINE (Life Technologies, Inc.), according to the manufacturer's instructions. Briefly, 20 μg of plasmid DNA is premixed with 80 μl of LipofectAMINE reagent, and this mixture was used to transfect COS7 cells 18 h after the subculture into 10 each of 140-mm dishes. At the time of transfection, the cell density was ∼25–30% confluent. After the transfection procedure in Opti-MEM for 5 h, α-minimum essential medium with 20% fetal calf serum was added to a final of 10%, followed by an overnight incubation in α-minimum essential medium. Transfected cells were washed with phosphate-buffered saline, scraped off from the dish, washed twice with phosphate-buffered saline, and homogenized in 25 mm Tris-HCl, pH 7.3, containing 1 mm dithiothreitol using gentle sonication. The homogenate was centrifuged 500 × g for 5 min, and the post-nuclear supernatant was then ultracentrifuged at 100,000 ×g for 40 min. The resulting supernatant was then incubated with anti-FLAG M2-Sepharose (Eastman-Kodak) resin overnight at 4 °C with rotation. The M2 resin was packed into a mini-column and eluted with 100 mm glycine-HCl, pH 3.0. The eluted fractions were neutralized immediately by adding drops of 1 m Tris-HCl, pH 8.5, and aliquots of each were subjected to 12.5% SDS-PAGE in either reduced or nonreduced conditions. Gels were then electrotransferred to nitrocellulose membranes (Bio-Rad) in a wet condition and blocked in 2% dry milk in Tris-buffered saline with 0.2% Tween. The blot was exposed to the primary anti-FLAG antibody M2 (1:2500 in Tris-buffered saline) for 1 h followed by washing and exposure to the secondary goat anti-mouse IgG horseradish peroxidase (Bio-Rad) for 1 h. Membranes were washed for more than 1 h. Horseradish peroxidase activity was then developed using the Supersignal substrate (Pierce) for 5 min. The signal was visualized by detection of chemiluminescence using x-ray film (Kodak) for periods ranging from a few seconds to 10 min. COS7 cells were transfected with FLAG-tagged constructs in 8-well chamber slides as described above. After overnight culture, cells were fixed with ice-cold acetone:methanol 1:1 and subjected to staining (the signal following 2 days of culture was too strong for the analysis). Briefly, fixed cells were washed with phosphate-buffered saline, incubated with 10 μg/ml of anti-FLAG antibody M2 (Eastman-Kodak) for 30 min at room temperature. Unbound antibody was washed off, and the cells were incubated with 1 μg/ml of fluorescein isothiocyanate-conjugated goat anti-mouse IgG for 30 min at room temperature. Unbound antibody was again washed off, and the slides were coverslipped using 9:1 glycerol/phosphate-buffered saline for viewing with a Zeiss Axiophot epifluoresence microscope. Images were captured using Adobe Photoshop software with a Sony CCD camera. Total cellular RNA was purified from various tissues of C57Bl/6 strain mice using Trizol reagent (Life Technologies, Inc.). Total RNA (20 μg) was reverse transcribed by superscript II (Life Technologies, Inc.) in a 20-μl reaction. 2.5 μl of first strand cDNA was used for PCR with 30 cycles for Lse amplification and 40 cycles for Cse-C amplification (preincubation at 94 °C for 2 min, 94 °C for 0.5 min, 60 °C for 1 min, and 72 °C for 2 min) using a Gene Amp 2400 (Perkin-Elmer). Primer sequences for the amplification were carried out using Lse-1 (anneals specific to Lse cDNA) versus Lse-J (should anneal to both forms) for Lse amplification and Cse-11 (anneals specific to Cse-C cDNA) versus Lse-J for Cse-C amplification. Sequence and position of these primers are as follows, Lse-1 (sense primer for Lse corresponding to positions cDNA 2–23) 5′-atcaggatcttcacaaacatggt-3′, Lse-J (antisense primer for Lse/Cse-C corresponding to Lse cDNA positions 816–795) and 5′-gaaacctctcttctggacaag-3′, and Cse-11 (sense primer for Cse-C corresponding to cDNA positions 124–144) 5′-aaaggacatgaggactcctcac-3′. Predicted amplified fragments were 810 bp for Lse and 890 bp for Cse-C (more than one band including 890 bp was actually seen for the latter). RT-PCR products were then directly sequenced or subcloned into a TA-cloning vector pCR II (Invitrogen), and multiple clones were sequenced by the cycle dideoxy termination sequencing method with an ABI model 310 autosequencer using M13–20 forward or reverse primers as primers. Not all the bands shown in agarose gel were recovered after gel purification, possibly because some are actually heterogenously annealed DNAs of different size. We had previously found that rabbits produced very low titer antibodies against intact Lse (33Butor C. Higa H.H. Varki A. J. Biol. Chem. 1993; 268: 10207-10213Abstract Full Text PDF PubMed Google Scholar). We therefore decided to raise antibodies against a defined peptide region in chickens. The 69-amino acid portion shared by Lse and Cse-C cDNAs (see Fig.1 B) was amplified using Pwo polymerase (Boehringer Mannheim) and the product subcloned into pMAL-p2 vector (New England Biolabs) as an in-frame maltose-binding protein fusion protein. The fusion protein was induced in LB containing 0.3 mm isopropyl 1-thio-β-d-galactoside at 37 °C for 3 h. Secreted fusion protein was directly purified from the medium by passage through a maltose column. The purity of the purified protein was confirmed by Coomassie Brilliant Blue staining after SDS-PAGE. The purified protein was used to immunize Rhode Island Red female chickens four times over a period of 2 months. After 2 months from the initial immunization, nonfertile eggs were collected, and the IgY was purified from egg yolk by using the Promega Eggstract kit. Mouse (C57Bl/6) liver was subjected to three cycles of freeze and thaw in hypotonic buffer to release the soluble lysosomal enzymes. The mixtures were then homogenized on ice with a polytron for a total of 2 min with intervals of 30 s on ice. The homogenates were ultracentrifuged at 100,000 × g for 40 min, and the supernatant fractions were then subjected to column chromatography on ConA-Sepharose (Amersham Pharmacia Biotech). The bound fractions were eluted with 100 mm α-methylmannopyranoside in 20 mm KPO4 pH 8, 0.1 m NaCl. ConA run-through fractions positive for Sia 9-O-acetylesterase activity (see assay described below) were pooled, dialyzed against 20 mm KPO4 pH 8, and loaded onto a DE52 (DEAE-cellulose) column (Whatman). After washing, the bound proteins were eluted with 15 ml of 150–400 mm NaCl gradient, followed by 5 ml of 400 mm NaCl (36Higa H.H. Manzi A. Varki A. J. Biol. Chem. 1989; 264: 19435-19442Abstract Full Text PDF PubMed Google Scholar). 25 μl each of the fractions from the ConA and DE52 columns were subjected to 12.5% SDS-PAGE under reduced conditions as described above. Transferred protein blots were incubated with anti-esterase C12 IgY antibody as the primary antibody, followed by development with a peroxidase-conjugated donkey anti-chicken IgY secondary antibody (Bio-Rad) and Supersignal substrate (Pierce). As described previously (43Higa H.H. Manzi A. Diaz S. Varki A. Methods Enzymol. 1989; 179: 409-415Crossref PubMed Scopus (6) Google Scholar), [9-O-acetyl-3H]Neu5,9Ac2 is prepared by labeling purified rat liver Golgi with [acetyl-3H]acetyl coenzyme A. The enzyme source (50–100 μl) is incubated with the substrate (10,000 cpm) at 37 °C for 1 h, and the reaction is quenched by the addition of equal volume of “stopping mixture,” followed by mixing with a toluene-based scintillation mixture. The uncleaved substrate cannot enter the toluene phase, but free [3H]Acetate (the cleavage product) can, allowing determination of the activity. Aliquots of fractions positive for the activity/Western blot signal for the chicken antibody were incubated with PNGase F for the cleavage of N-linked oligosaccharides. The digested material was compared with sham-treated material and analyzed by Western blotting. During characterization of the cDNA for the lysosomal Sia 9-O-acetylesterase (Lse) (39Stoddart A. Zhang Y. Paige C.J. Nucleic Acids Res. 1996; 24: 4003-4008Crossref PubMed Scopus (21) Google Scholar), we cloned a novel cDNA from a pre B cell line library (70Z/3), in which the signal peptide encoding region of Lse in its 5′ region was substituted with a novel sequence (Fig. 1). Because the substituted region completely matches exon 1 of the Lse gene, 2H. Takematsu and A. Varki, unpublished data. this new message seemed likely to be derived from an alternate promoter usage. The new cDNA is missing the original ATG codon of the Lse but has multiple in-frame ATG codons throughout the substituted 5′ region and the common sequence. Because the consensus sequence for endoplasmic reticulum targeting (the signal peptide) would be missing from the putative polypeptide encoded by this cDNA, we considered it a candidate for the previously described cytosolic Cse activity; this cDNA is hereafter called the Cse candidate (Cse-C). To determine the functional open reading frame of Cse-C, we created constructs for transient expression of Cse-C and the original Lse, each with a FLAG epitope tag incorporated into its C terminus. Anti-FLAG affinity chromatography was used to isolate epitope-tagged proteins from COS7 cells transiently transfected with either Lse-FLAG or Cse-C-FLAG. Production of polypeptides were studied by Western blotting using the anti-FLAG M2 antibody. Most of the Lse protein was recovered as a secreted form in the medium. This is in keeping with previous reports that a significant fraction of lysosomal enzymes are secreted into the medium when hyperexpressed in COS cells (38Guimaraes M.J. Bazan J.F. Castagnola J. Diaz S. Copeland N.G. Gilbert D.J. Jenkins N.A. Varki A. Zlotnik A. J. Biol. Chem. 1996; 271: 13697-13705Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In contrast, the Cse-C-FLAG product was mainly recovered in the cell lysate. Western blotting analysis of the anti-FLAG-Sepharose eluate showed that the secreted Lse was the expected uncleaved single band in the medium (the apparent molecular mass of 82 kDa is higher than that of the polypeptide backbone, presumably because of N-glycosylation and possibly O-glycosylation; see below). The Cse-C-FLAG product purified from cell lysates showed two bands of 51.5 and 28 kDa under reducing conditions (Fig.2 A). The smaller 28-kDa band showed a slower mobility in nonreducing conditions, indicating that the Cse-C-FLAG product can be cleaved by an endogenous proteinase to form heteromeric subunits (data not shown). Assuming that the 28-kDa fragment originated from the 51.5-kDa pro-polypeptide and accounting for the Kozak sequences (44Kozak M. J. Cell Biol. 1991; 115: 887-903Crossref PubMed Scopus (1452) Google Scholar) required for a functional starting codon (Fig. 2 B), it is likely that the translation machinery in COS7 cells is using the Met-75 ATG downstream from the N terminus of the Lse open reading frame to create a protein missing the signal sequence and some of the N-terminal region (a total of 75 amino acids is truncated, compared with the mature Lse). The comparison between the Lse and putative Cse-C sequences is shown in Fig. 1 B. The FLAG epitope-tagged forms of both molecules showed Sia 9-O-acetylesterase activity when affinity purified with anti-FLAG-Sepharose and assayed (data not shown). However, the Cse-C activity was rather unstable to routine elution with low pH. We therefore eluted the beads with a more gentle method, using the FLAG peptide itself (Fig. 2 C). The activity recovered by this method was also not completely stable, falling by about half during 1 week of storage at 4 °C. In contrast, the Lse-FLAG product showed activity that was stable to acid elution, as well as prolonged storage at 4 °C (data not shown). These properties are in keeping with those previously reported for lysosomal and cytosolic Sia 9-O-acetylesterase activities from biological sources (see “Discussion”). Because of the small amount of the Cse-C-FLAG product and activity available from the cell lysate and the instability to storage, more detailed studies of its enzymatic activity were not pursued. To confirm that the Cse-C encodes a cytosolic protein, we again transfected COS7 cells with the Lse-FLAG and Cse-C-FLAG constructs and detected the subcellular localization of