Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Genes Modulated by Expression of GD3 Synthase in Chinese Hamster Ovary Cells

2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês

10.1074/jbc.m210565200

ISSN

1083-351X

Autores

Honoo Satake, Helen Y. Chen, Ajit Varki,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

9-O-Acetylation is a common sialic acid modification, expressed in a developmentally regulated and tissue/cell type-specific manner. The relevant 9-O-acetyltransferase(s) have not been isolated or cloned; nor have mechanisms for their regulation been elucidated. We previously showed that transfection of the GD3 synthase (ST8Sia-I) gene into Chinese hamster ovary (CHO)-K1 cells gave expression of not only the disialoganglioside GD3 but also 9-O-acetyl-GD3. We now use differential display PCR between wild type CHO-K1 cells and clones stably expressing GD3 synthase (CHO-GD3 cells) to detect any increased expression of other genes and explore the possible induction of a 9-O-acetyltransferase. The four CHO mRNAs showing major up-regulation were homologous to VCAM-1, Tis21, the KC-protein-like protein, and a functionally unknown type II transmembrane protein. A moderate increase in expression of the FxC1 and SPR-1 genes was also seen. Interestingly, these are different from genes observed by others to be up-regulated after transfection of GD3 synthase into a neuroblastoma cell line. We also isolated a CHO-GD3 mutant lacking 9-O-acetyl-GD3 following chemical mutagenesis (CHO-GD3-OAc−). Analysis of the above differential display PCR-derived genes in these cells showed that expression of Tis21 was selectively reduced. Transfection of a mouseTis21 cDNA into the CHO-GD3-OAc− mutant cells restored 9-O-acetyl-GD3 expression. Since the only major gangliosides expressed by CHO-GD3 cells are GD3 and 9-O-acetyl-GD3 (in addition to GM3, the predominant ganglioside type in wild-type CHO-K1 cells), we conclude that GD3 enhances its own 9-O-acetylation via induction of Tis21. This is the first known nuclear inducible factor for 9-O-acetylation and also the first proof that 9-O-acetylation can be directly regulated by GD3 synthase. Finally, transfection of CHO-GD3-OAc− mutant cells with ST6Gal-I induced 9-O-acetylation specifically on sialylatedN-glycans, in a manner similar to wild-type cells. This indicates separate machineries for 9-O-acetylation on α2–8-linked sialic acids of gangliosides and on α2–6-linked sialic acids on N-glycans. 9-O-Acetylation is a common sialic acid modification, expressed in a developmentally regulated and tissue/cell type-specific manner. The relevant 9-O-acetyltransferase(s) have not been isolated or cloned; nor have mechanisms for their regulation been elucidated. We previously showed that transfection of the GD3 synthase (ST8Sia-I) gene into Chinese hamster ovary (CHO)-K1 cells gave expression of not only the disialoganglioside GD3 but also 9-O-acetyl-GD3. We now use differential display PCR between wild type CHO-K1 cells and clones stably expressing GD3 synthase (CHO-GD3 cells) to detect any increased expression of other genes and explore the possible induction of a 9-O-acetyltransferase. The four CHO mRNAs showing major up-regulation were homologous to VCAM-1, Tis21, the KC-protein-like protein, and a functionally unknown type II transmembrane protein. A moderate increase in expression of the FxC1 and SPR-1 genes was also seen. Interestingly, these are different from genes observed by others to be up-regulated after transfection of GD3 synthase into a neuroblastoma cell line. We also isolated a CHO-GD3 mutant lacking 9-O-acetyl-GD3 following chemical mutagenesis (CHO-GD3-OAc−). Analysis of the above differential display PCR-derived genes in these cells showed that expression of Tis21 was selectively reduced. Transfection of a mouseTis21 cDNA into the CHO-GD3-OAc− mutant cells restored 9-O-acetyl-GD3 expression. Since the only major gangliosides expressed by CHO-GD3 cells are GD3 and 9-O-acetyl-GD3 (in addition to GM3, the predominant ganglioside type in wild-type CHO-K1 cells), we conclude that GD3 enhances its own 9-O-acetylation via induction of Tis21. This is the first known nuclear inducible factor for 9-O-acetylation and also the first proof that 9-O-acetylation can be directly regulated by GD3 synthase. Finally, transfection of CHO-GD3-OAc− mutant cells with ST6Gal-I induced 9-O-acetylation specifically on sialylatedN-glycans, in a manner similar to wild-type cells. This indicates separate machineries for 9-O-acetylation on α2–8-linked sialic acids of gangliosides and on α2–6-linked sialic acids on N-glycans. CMP-Sia:Galβ1–4GlcNAc α2–6-sialyltransferase soluble chimeric CD22 conjugated with IgG1 Fc domain soluble chimeric influenza C hemagglutinin esterase fused to IgG1 Fc domain diisopropyl fluorophosphate-treated CHE-Fc Chinese hamster ovary CHO-K1 cells stably transfected with GD3 synthase gene 9-O-acetyl-GD3-deficient CHO-GD3 mutant cell derived from CHO-GD3 cells differential display PCR ethylmethane sulfonate reverse transcriptase-mediated PCR GD3 synthase or CMP-Sia:GM3 α2–8-sialyltransferase fluorescein isothiocyanate fluorescence-activated cell sorting mitogen-activated protein kinase/extracellular signal-regulated kinase kinase vascular cell adhesion molecule-1. Ganglioside nomenclature is based on the system of Svennerholm (95Svennerholm L. J. Neurochem. 1963; 10: 613-623Google Scholar) Sialic acids are a family of 9-carbon carboxylated monosaccharides typically located at the termini of mammalian cell surface sugar chains on both glycoproteins and glycolipids. N-acetylneuraminic acid, the most common sialic acid, is subjected to various modifications in vivo (1Schauer R. Cell Biol. Monogr. 1982; : 10Google Scholar, 2Varki A. Glycobiology. 1992; 2: 25-40Google Scholar, 3Kelm S. Schauer R. Int. Rev. Cytol. 1997; 175: 137-240Google Scholar, 4Varki A. FASEB J. 1997; 11: 248-255Google Scholar, 5Angata T. Varki A. Chem. Rev. 2002; 102: 439-470Google Scholar). One of the most prevalent modifications is O-acetylation of the hydroxyl group at the 9-carbon position. This modification is known to reduce or abolish the recognition of sialic acid residues by sialidases (2Varki A. Glycobiology. 1992; 2: 25-40Google Scholar, 4Varki A. FASEB J. 1997; 11: 248-255Google Scholar, 6Schauer R. Kelm S. Reuter G. Roggentin P. Shaw L. Rosenberg A. Biology of the Sialic Acids. Plenum Press, New York1995: 7-67Google Scholar, 7Reuter G. Gabius H.J. Biol. Chem. Hoppe-Seyler. 1996; 377: 325-342Google Scholar), by certain sialic acid-binding lectins like Siglecs (4Varki A. FASEB J. 1997; 11: 248-255Google Scholar, 5Angata T. Varki A. Chem. Rev. 2002; 102: 439-470Google Scholar, 8Kelm S. Schauer R. Manuguerra J.-C. Gross H.-J. Crocker P.R. Glycoconj. J. 1994; 11: 576-585Google Scholar, 9Sjoberg E.R. Powell L.D. Klein A. Varki A. J. Cell Biol. 1994; 126: 549-562Google Scholar, 10Shi W.X. Chammas R. Varki N.M. Powell L. Varki A. J. Biol. Chem. 1996; 271: 31526-31532Google Scholar), and by several viral recognition proteins (4Varki A. FASEB J. 1997; 11: 248-255Google Scholar, 5Angata T. Varki A. Chem. Rev. 2002; 102: 439-470Google Scholar, 11Rogers G.N. Paulson J.C. Virology. 1983; 127: 361-373Google Scholar, 12Suzuki Y. Ito T. Suzuki T. Holland R.E.J. Chambers T.M. Kiso M. Ishida H. Kawaoka Y. J. Virol. 2000; 74: 11825-11831Google Scholar). Conversely, other viruses require 9-O-acetylation for recognition of their target cells (4Varki A. FASEB J. 1997; 11: 248-255Google Scholar, 5Angata T. Varki A. Chem. Rev. 2002; 102: 439-470Google Scholar, 13Rogers G.N. Herrler G. Paulson J.C. Klenk H.D. J. Biol. Chem. 1986; 261: 5947-5951Google Scholar, 14Harms G. Reuter G. Corfield A.P. Schauer R. Glycoconj. J. 1996; 13: 621-630Google Scholar, 15Vlasak R. Luytjes W. Spaan W. Palese P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4526-4529Google Scholar, 16Schultze B. Herrler G. Arch. Virol. 1994; 136: 451-459Google Scholar). It is also known that 9-O-acetylation is regulated during development and aberrantly expressed in melanomas and basal cell carcinomas (17Cheresh D.A. Reisfeld R.A. Varki A. Science. 1984; 225: 844-846Google Scholar, 18Sparrow J.R. Barnstable C.J. J. Neurosci. 1988; 21: 398-409Google Scholar, 19Heidenheim M. Hansen E.R. Baadsgaard O. Br. J. Dermatol. 1995; 133: 392-397Google Scholar, 20Fahr C. Schauer R. J. Invest. Dermatol. 2001; 116: 254-260Google Scholar). These findings indicate diverse physiological and pathological roles for 9-O-acetylation of sialic acid residues. O-Acetyl groups can be added to the 7- and/or 9-position of sialic acids, with the former migrating to the 9-position, either spontaneously under physiological conditions (21Varki A. Diaz S. Anal. Biochem. 1984; 137: 236-247Google Scholar, 22Kamerling J.P. Schauer R. Shukla A.K. Stoll S. van Halbeek H. Vliegenthart J.F.G. Eur. J. Biochem. 1987; 162: 601-607Google Scholar) or under the influence of a specific migrase enzyme (23Vandamme-Feldhaus V. Schauer R. J. Biochem. (Tokyo). 1998; 124: 111-121Google Scholar). In the previous studies, we and others showed that 9(7)-O-acetylation is an acetyl-CoA-dependent enzymatic reaction (24Schauer R. Wember M. Hoppe-Seyler's Z Physiol. Chem. 1971; 352: 1282-1290Google Scholar, 25Corfield A.P. Ferreira D.A.C. Wember M. Schauer R. Eur. J. Biochem. 1976; 68: 597-610Google Scholar, 26Higa H.H. Butor C. Diaz S. Varki A. J. Biol. Chem. 1989; 264: 19427-19434Google Scholar, 27Butor C. Diaz S. Varki A. J. Biol. Chem. 1993; 268: 10197-10206Google Scholar, 28Shen Y. Tiralongo J. Iwersen M. Sipos B. Kalthoff H. Schauer R. Biol. Chem. 2002; 383: 307-317Google Scholar) that appears to be localized in the trans-Golgi apparatus (26Higa H.H. Butor C. Diaz S. Varki A. J. Biol. Chem. 1989; 264: 19427-19434Google Scholar,27Butor C. Diaz S. Varki A. J. Biol. Chem. 1993; 268: 10197-10206Google Scholar, 29Sjoberg E.R. Manzi A.E. Khoo K.-H. Dell A. Varki A. J. Biol. Chem. 1992; 267: 16200-16211Google Scholar, 30Chammas R. McCaffery J.M. Klein A. Ito Y. Saucan L. Palade G. Farquhar M.G. Varki A. Mol. Biol. Cell. 1996; 7: 1691-1707Google Scholar) and is presumably catalyzed by a sialic acid-specific 9(7)-O-acetyltransferase. Available evidence also suggests that there are multiple distinct O-acetyltransferases responsible for O-acetylating sialic acids attached to glycans in different linkages and possibly for different classes of glycan chains. For example, transfection of a cDNA encoding CMP-Sia:Galβ1–4GlcNAc α2–6-sialyltransferase (ST6Gal-I)1 into Chinese hamster ovary (CHO)-K1 cells (which normally express only α2–3-linked sialic acids) induced the expression of 9-O-acetyl groups only on the newly appearing α2–6-linked sialic acids of N-glycans (31Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 15130-15138Google Scholar). Likewise, transfection of GD3 synthase (CMP-Sia:GM3 α2–8-sialyltransferase (ST8Sia-I)) into CHO-K1 cells resulted in generation of 9-O-acetyl groups only on α2–8-linked sialic acids of the newly synthesized disialoganglioside GD3 (31Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 15130-15138Google Scholar). In contrast, no 9-O-acetyl sialic acids were detected upon transfection of CMP-Sia:Galβ1–3(4)GlcNAc α2–3 sialyltransferase into CHO-K1 cells (31Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 15130-15138Google Scholar). On the other hand, regulated 9-O-acetylation on murine erythroleukemia cells and on murine T cells appears to be on mucin-like molecules, presumably carried on O-glycans (32Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 31517-31525Google Scholar, 33Krishna M. Varki A. J. Exp. Med. 1997; 185: 1997-2013Google Scholar). Many groups have attempted to isolate or clone the 9-O-acetyltransferases responsible for these phenomena, with no success so far. The 9-O-acetyltransferase activity in most systems is very sensitive to solubilization, and thus, direct purification has proven difficult (27Butor C. Diaz S. Varki A. J. Biol. Chem. 1993; 268: 10197-10206Google Scholar, 28Shen Y. Tiralongo J. Iwersen M. Sipos B. Kalthoff H. Schauer R. Biol. Chem. 2002; 383: 307-317Google Scholar, 34Diaz S. Higa H.H. Hayes B.K. Varki A. J. Biol. Chem. 1989; 264: 19416-19426Google Scholar). Heterologous cell-cDNA library pairs were applied by several investigators to the attempted expression cloning of a 9-O-acetyltransferase cDNA in COS cells (35Ogura K. Nara K. Watanabe Y. Kohno K. Tai T. Sanai Y. Biochem. Biophys. Res. Commun. 1996; 225: 932-938Google Scholar, 36Kanamori A. Nakayama J. Fukuda M.N. Stallcup W.B. Sasaki K. Fukuda M. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2897-2902Google Scholar, 37Shi W.X. Chammas R. Varki A. Glycobiology. 1998; 8: 199-205Google Scholar). However, whereas several candidate genes were isolated, neither a 9-O-acetyltransferase nor a biologically significant inducer for 9-O-acetylation was eventually defined. This suggests that the 9-O-acetyltransferase could be a complex of multiple gene products, not amenable to standard expression cloning. This would fit with our proposal in rat liver Golgi that the acetyl group is transferred to luminal sialic acids via a transmembrane acetyl transfer reaction (26Higa H.H. Butor C. Diaz S. Varki A. J. Biol. Chem. 1989; 264: 19427-19434Google Scholar, 34Diaz S. Higa H.H. Hayes B.K. Varki A. J. Biol. Chem. 1989; 264: 19416-19426Google Scholar). The induction of 9-O-acetyl sialic acid by transfection of ST8Sia-I (GD3 synthase) into wild CHO-K1 cells described above can be explained either by the up-regulation of the 9-O-acetyltransferase gene in the presence of this sialyltransferase or by the preexisting expression of an α2–8 linkage-specific 9-O-acetyltransferase gene in CHO-K1 cells. If the former is true, analysis of gene expression differences between wild-type CHO-K1 and sialyltransferase gene-transfected CHO cells offers the possibility of detecting the putative 9-O-acetyltransferase gene and/or biologically significant inducers of 9-O-acetylation. We have therefore performed differential display PCR (DD-PCR) between wild type CHO-K1 cells and CHO-K1 cells stably transfected with ST8Sia-I (CHO-GD3 cells). In doing so, we also intended to detect other interesting genes up-regulated by the expression of ST8Sia-I. Such genes might be of interest regarding the induction of 9-O-acetylation or the involvement of GD3 and/or GD3 synthase in biological functions such as differentiation, tissue organization, and regeneration (38Varki A. Hooshmand F. Diaz S. Varki N.M. Hedrick S.M. Cell. 1991; 65: 65-74Google Scholar, 39Novikov A.M. Seyfried T.N. Biochem. Genet. 1991; 29: 627-638Google Scholar, 40Mendez-Otero R. Cavalcante L.A. Neurosci. Lett. 1996; 204: 97-100Google Scholar, 41Yamamoto A. Yamashiro S. Fukumoto S. Haraguchi M. Atsuta M. Shiku H. Furukawa K. Glycoconj. J. 1996; 13: 471-480Google Scholar, 42Liu H. Kojima N. Kurosawa N. Tsuji S. Glycobiology. 1997; 7: 1067-1076Google Scholar, 43Kawai H. Sango K. Mullin K.A. Proia R.L. J. Biol. Chem. 1998; 273: 19634-19638Google Scholar, 44Birklé S. Gao L.Y. Zeng G.C. Yu R.K. J. Neurochem. 2000; 74: 547-554Google Scholar, 45Panzetta P. Allende M.L. Neurochem. Res. 2000; 25: 163-169Google Scholar, 46Santiago M.F. Berredo-Pinho M. Costa M.R. Gandra M. Cavalcante L.A. Mendez-Otero R. Mol. Cell. Neurosci. 2001; 17: 488-499Google Scholar, 47Okada M. Itoh M. Haraguchi M. Okajima T. Inoue M. Oishi H. Matsuda Y. Iwamoto T. Kawano T. Fukumoto S. Miyazaki H. Furukawa K. Aizawa S. J. Biol. Chem. 2002; 277: 1633-1636Google Scholar), tumor biology (19Heidenheim M. Hansen E.R. Baadsgaard O. Br. J. Dermatol. 1995; 133: 392-397Google Scholar, 20Fahr C. Schauer R. J. Invest. Dermatol. 2001; 116: 254-260Google Scholar,48Dippold W.G. Dienes H.P. Knuth A. Meyer Z.B.K.H. Cancer Res. 1985; 45: 3699-3705Google Scholar, 49Ye J.N. Gu T. Xia L. Murphy M.J.J. Lee W. Gao N. Gu J. Fuhrer J.P. Cancer Res. 1990; 50: 7697-7702Google Scholar), and apoptosis (50De M.R. Lenti L. Malisan F. D'Agostino F. Tomassini B. Zeuner A. Rippo M.R. Testi R. Science. 1997; 277: 1652-1655Google Scholar, 51Kristal B.S. Brown A.M. J. Biol. Chem. 1999; 274: 23169-23175Google Scholar, 52Scorrano L. Petronilli V. Di L.F. Bernardi P. J. Biol. Chem. 1999; 274: 22581-22585Google Scholar, 53Farina F. Cappello F. Todaro M. Bucchieri F. Peri G. Zummo G. Stassi G. J. Histochem. Cytochem. 2000; 48: 57-62Google Scholar, 54Rippo M.R. Malisan F. Ravagnan L. Tomassini B. Condo I. Costantini P. Susin S.A. Rufini A. Todaro M. Kroemer G. Testi R. FASEB J. 2000; 14: 2047-2054Google Scholar, 55Giammarioli A.M. Garofalo T. Sorice M. Misasi R. Gambardella L. Gradini R. Fais S. Pavan A. Malorni W. FEBS Lett. 2001; 506: 45-50Google Scholar, 56Copani A. Melchiorri D. Caricasole A. Martini F. Sale P. Carnevale R. Gradini R. Sortino M.A. Lenti L. De M.R. Nicoletti F. J. Neurosci. 2002; 22: 3963-3968Google Scholar). Using this approach and a 9-O-acetyl-GD3-deficient CHO-GD3 mutant cell (CHO-GD3-OAc−), we present evidence for a novel pathway in which GD3 induces its own 9-O-acetylation via up-regulation of Tis21, a member of an antiproliferative protein family. Unless otherwise stated, reagents were purchased from Fisher or Sigma, and all oligonucleotides were from Invitrogen. CHO-K1 cells were obtained from the American Type Culture Collection (ATCC CCL61). All CHO cell lines were grown under 5% CO2 and 100% relative humidity in α-minimum essential medium supplemented with 10% (v/v) fetal bovine serum. The mouse IgG3 anti-GD3 monoclonal antibody R24 (57Pukel C.S. Lloyd K.O. Travassos L.R. Dippold W.G. Oettgen H.F. Old L.J. J. Exp. Med. 1982; 155: 1133-1147Google Scholar) was purified from hybridoma supernatant using Protein A-Sepharose as previously described (58Chammas R. Sonnenburg J.L. Watson N.E. Tai T. Farquhar M.G. Varki N.M. Varki A. Cancer Res. 1999; 59: 1337-1346Google Scholar). Biotinylated R24 was generated by incubating 20 μg of the antibody with 4 μg of EZ-link sulfo-N-hydroxysuccinimide-biotin (Pierce) in 0.1m NaHCO3 (pH 8.3) for 2 h at 4 °C, followed by dialysis against phosphate-buffered saline to remove the excess reagent. The mouse IgG3 anti-9-O-acetyl-GD3 monoclonal antibody 27A (59Reivinen J. Holther H. Miettinen A. Kidney Int. 1992; 42: 624-631Google Scholar) was kindly provided by Dr. M. Farquhar (University of California, San Diego) and was used as hybridoma supernatant, or it was purified from supernatant using Protein A-Sepharose (Amersham Biosciences) following the manufacturer's instructions. FITC-conjugated 27A was generated using a Fluorotag FITC Conjugation Kit (Sigma) following the manufacturer's instructions. A recombinant soluble chimera of influenza C hemagglutinin-esterase fused to the Fc region of human IgG1 (CHE-Fc) (60Klein A. Krishna M. Varki N.M. Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7782-7786Google Scholar) was produced by stably transfected HEK-293 cells. These cells were also adapted to a protein-free medium, CHO-S-SFM II, and generously provided by Dr. Pascal Crottet (University of Basel) (61Dumermuth E. Beuret N. Spiess M. Crottet P. J. Biol. Chem. 2002; 277: 18687-18693Google Scholar). CHE-FcD, the esterase-inactive form of CHE-Fc (60Klein A. Krishna M. Varki N.M. Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7782-7786Google Scholar), was prepared by treatment with diisopropyl fluorophosphate as previously described (60Klein A. Krishna M. Varki N.M. Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7782-7786Google Scholar). Conditioned medium was collected and concentrated to 4 μg/ml protein concentration. For each staining, 150 μl of CHE-FcD-containing medium was precomplexed with phycoerythrin-conjugated anti-human IgG1 at a 1:15 dilution (preoptimized for this batch of antibody) in the dark for 2 h at 4 °C. The antibodies were then preabsorbed with CHO-K1 wild type cells to eliminate nonspecific cell surface binding. Samples were stained with these preabsorbed CHE-FcD preparations for 2 h at 4 °C and analyzed by flow cytometry. CD22-Fc, the soluble chimera of the extracellular domain of CD22 fused to the human IgG1 Fc domain, was purified and used at 5 μg/ml concentration for cell staining, as previously described (62Brinkman-Van der Linden E.C.M. Sjoberg E.R. Juneja L.R. Crocker P.R. Varki N. Varki A. J. Biol. Chem. 2000; 275: 8633-8640Google Scholar). The expression vector carrying ST8Sia-I for transient transfection has been reported (31Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 15130-15138Google Scholar). Stable expression of the same cDNA was obtained by transfecting a pcDNA3-based construct into parental CHO-K1 cells using the LipofectAMINE reagent (Invitrogen) and selected with G418. After 68 h, cells were stained with antibody 27A as described below, and individual positive cells were sorted into 96-well plates by fluorescence-activated cell sorting (FACS) using a FACStar unit (Becton Dickinson). The levels of 9-O-acetyl-GD3 expression of individual clones were analyzed by flow-cytometric analysis as described below. One clone that showed high and stable expression of 9-O-acetyl-GD3 (named CHO-GD3) was used for further experiments. The cloned CHO-GD3 cell line (stably transfected with ST8Sia-I) was incubated in α-minimum essential medium containing 450 μg/ml ethylmethane sulfonate (EMS; Sigma) for 16 h in a 37 °C incubator. The concentration of EMS was determined in pilot experiments in which the frequency of ouabain resistance was monitored, exactly as previously described (63Esko J.D. Methods Cell Biol. 1989; 32: 387-422Google Scholar). Cells were allowed to recover for 72 h in regular medium. Mutagenized cells were then stained with FITC-conjugated 27A antibody and with biotinylated R24 antibody followed by streptavidin-Cychrome (Pharmingen). Cells that stained positive for R24 and negative for 27A were isolated on a FACStar unit (Becton Dickinson). Individual clones were analyzed by flow cytometry as described below. One clone that showed stable GD3 expression (R24-positive) without detectable 9-O-acetylation (27A-negative) was expanded and named CHO-GD3-OAc−. Total RNA was extracted from CHO-K1 wild type and CHO-GD3, respectively, using RNeasy minikits (Qiagen) and reverse-transcribed using Moloney murine leukemia virus reverse transcriptase and oligo(dT11N) (where N represents A, C, or G) reverse primers (Genhunter) according to the manufacturer's instructions. The resultant first strand cDNA was amplified by PCR using 240 primer pairs of 80 arbitrary forward primers and three oligo(dT11N) primer sets (where N represents A, C, or G; Genhunter) and Amplitaq polymerase (PerkinElmer Life Sciences). PCR products were labeled by [33P]dATP (PerkinElmer Life Sciences) contained in the PCR mixture. The 33P-labeled PCR products were resolved on 6% acrylamide gels followed by extraction of the products that were specifically enhanced in CHO-GD3 cells. The isolated products were subcloned into a pCR2.1 TOPO vector (Invitrogen) and sequenced on both strands using M13 primers and an automated DNA sequencer (model 373A; PerkinElmer Life Sciences). First strand cDNA was synthesized from total RNA of CHO-K1 wild type, CHO-GD3, and CHO-GD3-OAc− using oligo(dT) primer and Superscript II (Invitrogen). PCR was performed using Amplitaq polymerase (PerkinElmer Life Sciences) and gene-specific primers for each DD-PCR product. In addition, the expression of the Chinese hamster isopentenyl-diphosphate: dimethylallyl diphosphate isomerase gene (GenBankTM accession number AF003836) was employed as a control. The RT-PCR products were subjected to electrophoresis on a 1.5% agarose gel. A mouse Tis21 open reading frame was prepared by PCR from a mouse 5′-expressed sequence tag clone (accession number BG973699) using a forward primer (5′-CTGTAGAATTCGCCATGAGCCACGGGAAGAGAAC-3′) and a reverse primer (5′-ATAAGAATGCGGCCGCCAGCTGGAGACGGCCATCACAT-3′). The PCR product was digested with EcoRI and NotI followed by ligation into a EcoRI/NotI site of an expression vector, pcDNA3.1myc-His A (Invitrogen). The sequence of the subcloned insert was confirmed by sequencing of PCR products obtained using T7 forward and BGH reverse primers. Mouse Tis21-containing pcDNA 3.1 plasmid or empty vector was transfected into CHO-GD3-OAc− using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions, and the cells were incubated for 48 h. The cycle of transfection, 48-h incubation, and detachment from a culture plate was carried out three times, and re-expression of 9-O-acetyl-GD3 was monitored by flow cytometry using 27A. Human ST6Gal-I-containing pcDNA 3.1 plasmid or empty vector was transfected into wild-type, CHO-GD3, or CHO-GD3-OAc−mutant cells using LipofectAMINE Plus reagent (Invitrogen), and the cells were incubated for 48 h. Cells were detached by EDTA solution (without trypsin treatment) and subjected to flow cytometric analysis using CHE-FcD to detect 9-O-acetyl groups. Various types of CHO cells (1 × 106) were trypsinized to expose gangliosides (or released with EDTA only when studying glycoproteins) and washed with phosphate-buffered saline containing 1% bovine serum albumin. The cells were then incubated with either R24 antibody (for detecting GD3), 27A antibody (for detecting 9-O-acetyl-GD3), CD22-Fc (for detecting α2–6-linked sialic acids without 9-O-acetylation), or CHE-FcD (for detecting 9-O-acetyl sialic acids). The reagents were either precomplexed or complexed in situ with phycoerythrin-conjugated goat anti-mouse secondary antibody as previously described. After final washing, the fluorescent intensity on the cell surface was observed on a Becton Dickinson FACScan instrument. Wild type CHO cells express predominantly the monosialoganglioside GM3 (64Weis F.M.B. Davis R.J. J. Biol. Chem. 1990; 265: 12059-12066Google Scholar, 65Lutz M.S. Jaskiewicz E. Darling D.S. Furukawa K. Young W.W.J. J. Biol. Chem. 1994; 269: 29227-29231Google Scholar, 66Warnock D.E. Lutz M.S. Blackburn W.A. Young W.W.J. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2708-2712Google Scholar). In our previous studies, we prepared CHO-K1 cells stably transfected with GD3 synthase (ST8Sia-I) (31Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 15130-15138Google Scholar). This cell population expressed not only disialoganglioside GD3 but also 9-O-acetyl-GD3 (detected with monoclonal antibodies R24 and 27A, respectively), neither of which are found in the wild type cells (31Shi W.X. Chammas R. Varki A. J. Biol. Chem. 1996; 271: 15130-15138Google Scholar). During continuous culture, we noticed that this population of cells expressed varying levels of GD3 and 9-O-acetyl-GD3 (data not shown). We therefore used FACS to isolate clones with consistently high expression of GD3 and 9-O-acetyl-GD3 (see example in Fig. 1). As expected, stable clones expressing GD3 synthase (called CHO-GD3) expressed primarily GD3, as determined by a resorcinol stain of high performance thin-layer chromatography-separated total gangliosides (not shown), with smaller amounts of 9-O-acetyl-GD3 (also detected in the total lipid fraction of cells labeled with [3H]galactose and studied by autoradiography; data not shown). Although the CHO-GD3 cells express 9-O-acetyl-GD3, neither these cells nor the wild-type CHO-K1 cells express 9-O-acetyl GM3. Thus, a GD3-specific 9-O-acetyltransferase must either preexist in wild-type CHO-K1 cells or be secondarily induced by the presence of GD3. (Since GD3 synthase is a Golgi-localized protein with a short cytoplasmic tail, it is unlikely that such induction is a direct effect of the synthase protein itself.) To explore these possibilities as well as to evaluate the effects of GD3 and 9-O-acetyl-GD3 on the expression of other genes, we performed DD-PCR between RNA preparations from CHO-K1 wild type and CHO-GD3 cells using 80 arbitrary primers and three oligo(dT11N) (where N represents A, C, or G) primers. To minimize artifacts and false positive bands, DD-PCR using all primer pairs was repeated three times. Six DD-PCR products that consistently showed differentially enhanced expression in CHO-GD3 cells were finally obtained. These products were extracted, reamplified, subcloned, and sequenced. We designated them as 19C, 26C, 38G, 44G, 64C, and 66G, according to the numbering of the arbitrary primers and oligo(dT11N) reverse primer sets originally used for detection of each. To confirm that message expression of these DD-PCR products was actually enhanced or induced in CHO-GD3, we performed RT-PCR using gene-specific primers for each product. As shown in Fig.2 A, 19C, 38G, 64C, and 66G were expressed in CHO-GD3 much more abundantly than in wild-type CHO-K1 cells. Expression of the other two products, 26C and 44G, was also increased in CHO-GD3 cells (Fig. 2 A). These results confirm that expression of all six mRNAs is up-regulated by stable transfection of the GD3 synthase gene. Similar data were obtained by comparative RT-PCR between wild-type cells and another CHO-GD3 clone, as well as with wild-type cells transiently transfected with GD3 synthase (Fig. 2 B). These results confirmed the consistent enhancement of expression of these genes by the presence of GD3. Nucleotide sequence analysis followed by BLAST homology searching of the NCBI data bases revealed similarities of each of the hamster DD-PCR products with other previously reported genes (see summary in TableI). Product 19C was most similar to the mouse Tis21/Btg2/PC3/APRO-1 gene (accession number M64292), which is a member of an anti-proliferative protein family (hereafter called the Tis21 gene) (67Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.A. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Google Scholar). The 64C sequence showed homology with mouse vascular cell adhesion molecule-1 (VCAM-1; accession number X67783) (68Araki M. Araki K. Vassalli P. Gene (Amst.). 1993; 126: 261-264Google Scholar). The 38G sequence was found to be homologous to a small type II transmembrane protein of unknown function (accession number AB015632) (69Yokoyama-Kobayashi M. Yamaguchi T. Sekine S. Kato S. Gene (Amst.). 1999; 228: 161-167Google Scholar). The 66G sequence is similar to a rat KC protein-like protein gen

Referência(s)