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• W2068746509 abstract "Poly-α-2,8-sialic acid (polysialic acid) is a post-translational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto- and NCAM polysialylation activity. Autopolysialylation occurs onN-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both auto- and NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends on specific N-glycans attached to Asn74 in ST8SiaIV and Asn89 and Asn219 in ST8SiaII. Deletion of polysialic acid acceptor sites by site-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylation-negative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions. Poly-α-2,8-sialic acid (polysialic acid) is a post-translational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto- and NCAM polysialylation activity. Autopolysialylation occurs onN-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both auto- and NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends on specific N-glycans attached to Asn74 in ST8SiaIV and Asn89 and Asn219 in ST8SiaII. Deletion of polysialic acid acceptor sites by site-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylation-negative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions. poly-α2,8-sialic acid neural cell adhesion molecule N-acetylneuraminic acid CMP-Neu5Ac-polysialosyl α-2,8-sialyltransferase II CMP-Neu5Ac-polysialosyl α-2,8-sialyltransferase IV endoneuraminidase NE monoclonal antibody peptide-N-glycosidase F YPYDVPDYA nonapeptide of the influenza virus hemagglutinin polymerase chain reaction base pair(s) Chinese hamster ovary polyacrylamide gel electrophoresis thymidine kinase-deficient murine fibroblasts Polysialic acid (PSA),1a linear homopolymer of α-2,8-linked sialic acids, is a unique post-translational modification of the neural cell adhesion molecule (NCAM) (1Mühlenhoff M. Eckhardt M. Gerardy-Schahn R. Curr. Opin. Struct. Biol. 1998; 8: 558-564Crossref PubMed Scopus (160) Google Scholar). PSA chains can be over 50 residues in length (2Livingston B.D. Jacobs J.L. Glick M.C. Troy F.A. J. Biol. Chem. 1988; 263: 9443-9448Abstract Full Text PDF PubMed Google Scholar, 3Inoue S. Lin S.L. Inoue Y. J. Biol. Chem. 2000; 275: 29968-29979Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and form helical secondary structures (4Brisson J.R. Baumann H. Imberty A. Perez S. Jennings H.J. Biochemistry. 1992; 31: 4996-5004Crossref PubMed Scopus (125) Google Scholar). Size and physicochemical properties of the PSA moiety profoundly influence the NCAM binding abilities (5Rutishauser U. Curr. Opin. Cell Biol. 1996; 8: 679-684Crossref PubMed Scopus (146) Google Scholar). Although NCAM mediates stable cell-cell contacts in the absence of PSA, the adhesion molecule is converted into an “anti-adhesive” factor in the presence of PSA (6Yang P. Major D. Rutishauser U. J. Biol. Chem. 1994; 269: 23039-23044Abstract Full Text PDF PubMed Google Scholar). PSA-NCAM is involved in promoting cell migration and axon guidance (7Hu H. Tomasiewicz H. Magnuson T. Rutishauser U. Neuron. 1996; 16: 735-743Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 8Rutishauser U. Landmesser L. Trends. Neurosci. 1996; 19: 422-427Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 9Murakami S. Seki T. Rutishauser U. Arai Y. J. Comp. Neurol. 2000; 420: 171-181Crossref PubMed Scopus (47) Google Scholar), and its expression during development was found to be highest in phases of neuronal motility (10Seki T. Arai Y. Neurosci. Res. 1991; 12: 503-513Crossref PubMed Scopus (183) Google Scholar). In the adult brain PSA is restricted to areas with persistent cellular plasticity such as hippocampus, hypothalamus, and olfactory bulb (11Bonfanti L. Olive S. Poulain D.A. Theodosis D.T. Neuroscience. 1992; 49: 419-436Crossref PubMed Scopus (300) Google Scholar, 12Theodosis D.T. Rougon G. Poulain D.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5494-5498Crossref PubMed Scopus (229) Google Scholar, 13Miragall F. Kadmon G. Faissner A. Antonicek H. Schachner M. J. Neurocytol. 1990; 19: 899-914Crossref PubMed Scopus (85) Google Scholar). Polysialylated NCAM represents an oncodevelopmental antigen (14Fukuda M. Cancer Res. 1996; 56: 2237-2244PubMed Google Scholar), and the re-expression of PSA was observed in several tumors. Recent studies strongly suggest that the PSA levels in these tumors correlate with their potential for metastasis (15Scheidegger E.P. Lackie P.M. Papay J. Roth J. Lab. Invest. 1994; 70: 95-106PubMed Google Scholar, 16Glüer S. Schelp C. Madry N. von Schweinitz D. Eckhardt M. Gerardy-Schahn R. Br. J. Cancer. 1998; 78: 106-110Crossref PubMed Scopus (39) Google Scholar, 17Daniel L. Trouillas J. Renaud W. Chevallier P. Gouvernet J. Rougon G. Figarella-Branger D. Cancer Res. 2000; 60: 80-85PubMed Google Scholar). Although a wide range of biological effects have been attributed to PSA, the biosynthesis of the sugar polymer and the regulation of its expression are understood poorly. Two polysialyltransferases, ST8SiaII (formerly named STX) and ST8SiaIV (formerly named PST-1 or PST), are able to synthesize PSA on NCAM. The genes of both enzymes have been cloned from diverse species (see Ref. 18Windfuhr M. Manegold A. Mühlenhoff M. Eckhardt M. Gerardy-Schahn R. J. Biol. Chem. 2000; 275: 32861-32870Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar and references cited therein), and their expression has been studied intensively during ontogenetic development (19Kurosawa N. Yoshida Y. Kojima N. Tsuji S. J. Neurochem. 1997; 69: 494-503Crossref PubMed Scopus (61) Google Scholar, 20Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Crossref PubMed Scopus (122) Google Scholar, 21Hildebrandt H. Becker C. Murau M. Gerardy-Schahn R. Rahmann H. J. Neurochem. 1998; 71: 2339-2348Crossref PubMed Scopus (95) Google Scholar). All studies describe ST8SiaII as the major isoform in embryonic and early postnatal brain, whereas ST8SiaIV was found to be the major isoform in adult brain. In line with this observation, inactivation of the ST8SiaIV gene by a knock-out mouse approach resulted in a nearly complete loss of PSA in mice older than 3 months and was found to be associated with severe deficits in synaptic plasticity (22Eckhardt M. Bukalo O. Chazal G. Wang L. Goridis C. Schachner M. Gerardy-Schahn R. Cremer H. Dityatev A. J. Neurosci. 2000; 20: 5234-5244Crossref PubMed Google Scholar). Both polysialyltransferases are individually able to polysialylate NCAM by adding PSA to monosialylated N-glycans (23Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. Curr. Biol. 1996; 6: 1188-1191Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The synthesis of PSA on NCAM is highly specific and involves only two of six N-glycosylation sites of NCAM (24Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 25Nelson R.W. Bates P.A. Rutishauser U. J. Biol. Chem. 1995; 270: 17171-17179Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). An interesting feature of the polysialyltransferases is their ability to catalyze autopolysialylation. During this autocatalytic process, PSA chains are build up on N-glycans of the enzymes (26Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. EMBO J. 1996; 15: 6943-6950Crossref PubMed Scopus (96) Google Scholar, 27Close B.E. Colley K.J. J. Biol. Chem. 1998; 273: 34586-34593Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Most recently autopolysialylation could be demonstrated also for ST8SiaIII, a sialyltransferase for which the natural acceptor substrate has not been identified thus far. Compared with ST8SiaII and ST8SiaIV, autocatalytically synthesized PSA chains were found to be shorter in ST8SiaIII (28Angata K. Suzuki M. McAuliffe J. Ding Y. Hindsgaul O. Fukuda M. J. Biol. Chem. 2000; 275: 18594-18601Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). When we initially described autopolysialylation, we also demonstrated that a secreted form of recombinant ST8SiaIV carried PSA (26Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. EMBO J. 1996; 15: 6943-6950Crossref PubMed Scopus (96) Google Scholar). Close and Colley (27Close B.E. Colley K.J. J. Biol. Chem. 1998; 273: 34586-34593Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) confirmed this observation and in addition showed that the full-length Golgi-localized polysialyltransferases ST8SiaII and -IV carry PSA. However, studies addressing the functional importance of this self-modification caused substantial controversy. Although data from our laboratory (18Windfuhr M. Manegold A. Mühlenhoff M. Eckhardt M. Gerardy-Schahn R. J. Biol. Chem. 2000; 275: 32861-32870Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 26Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. EMBO J. 1996; 15: 6943-6950Crossref PubMed Scopus (96) Google Scholar) suggest a tight mechanistic link between auto- and NCAM polysialylation, Close et al.(29Close B.E. Tao K. Colley K.J. J. Biol. Chem. 2000; 275: 4484-4491Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) recently described the two catalytic reactions to be independent. To clarify the role of autopolysialylation a large panel ofN-glycosylation variants of the polysialyltransferases ST8SiaII and -IV have been created by site-directed mutagenesis and used (i) to identify the number and positions of N-glycans involved in the autopolysialylation process and (ii) to investigate the functional abilities of N-glycosylation-deficient polysialyltransferases in vivo and in vitro. The detailed studies presented in this paper confirm our previous results that interference with the autopolysialylation process prevents the formation of active polysialyltransferases in vivo andin vitro. CMP-[14C]Neu5Ac (293 μCi/mmol) was purchased from Amersham Pharmacia Biotech.Endoneuraminidase NE (endoNE) was purified from the Escherichia coli K1 bacteriophage, PK1E, as described (30Gerardy-Schahn R. Bethe A. Brennecke T. Mühlenhoff M. Eckhardt M. Ziesing S. Lottspeich F. Frosch M. Mol. Microbiol. 1995; 16: 441-450Crossref PubMed Scopus (69) Google Scholar). The monoclonal antibodies (mAbs) KD11 directed against the transmembrane isoforms of NCAM (31Gerardy-Schahn R. Eckhardt M. Int. J. Cancer Suppl. 1994; 8: 38-42Crossref PubMed Scopus (21) Google Scholar) and mAb 735 directed against polysialic acid (32Frosch M. Görgen I. Boulnois G.J. Timmis K.N. Bitter-Suermann D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1194-1198Crossref PubMed Scopus (341) Google Scholar) were used after purification on Protein G and Protein A-Sepharose (Amersham Pharmacia Biotech), respectively. Recombinant peptide-N-glycosidase F (PNGaseF) and mAb 12CA5 directed against the hemagglutinin (HA) epitope (YPYDVPDYA) were purchased from Roche. mAb M5 directed against the Flag sequence (MDYKDDDDK) and biotinylated mAb SPA-27 directed against Protein A were obtained from Sigma. Recombinant soluble Protein A-NCAM was expressed in 2A10 cells and purified as described (26Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. EMBO J. 1996; 15: 6943-6950Crossref PubMed Scopus (96) Google Scholar). Rabbit anti-α-mannosidase II antiserum was a kind gift of Dr. K. Moremen (University of Georgia, Athens, GA) (33Moremen K.W. Touster O. Robbins P.W. J. Biol. Chem. 1991; 266: 16876-16885Abstract Full Text PDF PubMed Google Scholar, 34Velasco A. Hendricks L. Moremen K.W. Tulsiani D.R. Touster O. Farquhar M.G. J. Cell Biol. 1993; 122: 39-51Crossref PubMed Scopus (281) Google Scholar), and the eukaryotic expression vector pPROTA (35Sanchez-Lopez R. Nicholson R. Gesnel M.C. Matrisian L.M. Breathnach R. J. Biol. Chem. 1988; 263: 11892-11899Abstract Full Text PDF PubMed Google Scholar) was provided generously by Dr. R. Breathnach (INSERM, Nantes, France). The plasmids pFlagHA-ST8SiaII and pFlagHA-ST8SiaIV containing full-length cDNA of murine ST8SiaII and hamster ST8SiaIV, respectively, with the N-terminal Flag and HA tags were constructed as described previously (18Windfuhr M. Manegold A. Mühlenhoff M. Eckhardt M. Gerardy-Schahn R. J. Biol. Chem. 2000; 275: 32861-32870Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The plasmids pZeoSV-ST8SiaII and pZeoSV-ST8SiaIV contain the same coding sequence and were generated by subcloning the KpnI/XbaI fragment of pFlagHA-ST8SiaII and -IV into the vector pZeoSV (Invitrogen). The plasmids pPROTA-ST8SiaII and pPROTA-ST8SiaIV, containing a transin secretion signal sequence and the IgG binding domain of Staphylococcus aureus Protein A in front of truncated polysialyltransferases that lack their transmembrane domains, were constructed in the vector pPROTA (35Sanchez-Lopez R. Nicholson R. Gesnel M.C. Matrisian L.M. Breathnach R. J. Biol. Chem. 1988; 263: 11892-11899Abstract Full Text PDF PubMed Google Scholar). PCR products coding for Ser26–Thr375 of murine ST8SiaII and Arg27–Gln359 of hamster ST8SiaIV were amplified using the primer pairs 5′-TCTCAGAGATCGAAGAAG-3′/5′-TTACGTAGCCCCATCAC-3′ and 5′-CCAGAACTGAGGAGCAC-3′/5′-TTATTGCTTCATGCACTTCCC-3′, respectively. The vector pPROTA was linearized withEcoRI and filled in with Klenow polymerase, and PCR fragments were inserted by blunt-end ligation. During ligation theEcoRI site was destroyed in the case of pPROTA-ST8SiaII, whereas one EcoRI site at the 5′end of the ST8SiaIV coding sequence was retained in the case of pPROTA-ST8SiaIV. N-glycosylation sites were destroyed by substituting asparagine in the Asn-XXX-Ser/Thr motifs by glutamine. This was done by PCR using the QuikChangeTMsite-directed mutagenesis kit (Stratagene) following the guide lines given by the manufacturer. Mutagenic primers were designed to change the codons used for Asn (AAT or AAC) into a codon used for Gln (CAA or CAG) by two site-specific nucleotide exchanges. The sequences of all 22 primers are given in Table I. For site-directed mutations in theST8SiaII gene, the full-length murine cDNA subcloned in the vector pBluescript SK(−) (Stratagene) was used as a template in the PCRs. Amplified products containing the desired mutations were digested with EcoRI to generate a 952-bp fragment that was ligated into the EcoRI sites of pPROTA-ST8SiaII and pFlagHA-ST8SiaII, replacing the corresponding wild-type sequence. For the PCR-based mutations carried out in ST8SiaIV, a 930-bpEcoRI fragment from pPROTA-ST8SiaIV (bp 77–1006 of the coding sequence) subcloned into pBluescript SK(−) was used as a template. EcoRI fragments with site-specific mutations were subcloned into pPROTA-ST8SiaIV, and a 915-bpBsmBI/EcoRI fragment (bp 91–1006 of the coding sequence) was used for subcloning in pFlagHA-ST8SiaIV, replacing the corresponding wild-type sequence. The identity of all mutants both before and after subcloning was confirmed by dideoxy sequencing.Table IN-glycosylation mutants of the polysialyltransferases ST8SiaII and IVMutationNucleotide exchangeAmino acid exchangeMutation primersST8SiaIIΔ1178AAT → CAAN60Q5′-GAGCTGAAGTTGTAATCCAAGGCTCTTCACCGCCAGC-3′5′-GCTGGCGGTGAAGAGCCTTGGATTACAACTTCAGCTC-3′ST8SiaIIΔ2214AAT → CAAN72Q5′-GCTGTCGCTGACAGAAGTCAAGAAAGCCTTAAGCAC-3′5′-GTGCTTAAGGCTTTCTTGACTTCTGTCAGCGACAGC-3′ST8SiaIIΔ3265AAC → CAAN89Q5′-CCAAATGGAGACACCAACAGACGCTCTCTCTGAG-3′5′-CTCAGAGAGAGCGTCTGTTGGTGTCTCCATTTGG-3′ST8SiaIIΔ4400AAC → CAGN134Q5′-CGTGACAGCACAATGCAGGTGTCCCAGAACCTC-3′5′-GAGGTTCTGGGACACCTGCATTGTGCTGTCACG-3′ST8SiaIIΔ5655AAC → CAGN219Q5′-CCTTTGAGGACCTTGTGCAGGCCACGTGGCGGG-3′5′-CCCGCCACGTGGCCTGCACAAGGTCCTCAAAGG-3′ST8SiaIIΔ6700AAT → CAAN234Q5′-GCTGCACGGCCTCCAAGGGAGCATCCTGTGG-3′5′-CCACAGGATGCTCCCTTGGAGGCCGTGCAGC-3′ST8SiaIVΔ1148AAC → CAGN50Q5′-GTGTTTGAGCAGATCACTTGTGCAGAGCTCTGATAAAATCATTCG-3′5′-CGAATGATTTTATCAGAGCTCTGCACAAGTGATCTGCTCAAACAC-3′ST8SiaIVΔ2220AAT → CAGN74Q5′-GGCTGGAGAATCCAGTCTTCTTTAGTCC-3′5′-GGACTAAAGAAGACTGGATTCTCCAGCC-3′ST8SiaIVΔ3355AAT → CAGN119Q5′-CAGACGCCGGACGCTGCAGATTTCCCATGATCTGCAC-3′5′-GTGCAGATCATGGGAAATCTGCAGCGTCCGGCGTCTG-3′ST8SiaIVΔ4610AAT → CAAN204Q5′-GCATTTGGAGGCTTTCGGCAAGAGAGTGACAGAGC-3′5′-GCTCTGTCACTCTCTTGCCGAAAGCCTCCAAATGC-3′ST8SiaIVΔ5655AAT → CAGN219Q5′-GCATAGACTTTCCATGCTGCAGGACAGTGTCCTTTGG-3′5′-CCAAAGGACACTGTCCTGCAGCATGGAAAGTCTATGC-3′N-Glycosylation sites of ST8SiaII and ST8SiaIV were deleted by site-directed mutagenesis in a PCR approach using the mutation primers given in this table. The codon used for asparagine in the Asn-Xxx-Ser/Thr motif was changed to glutamine by the indicated nucleotide exchanges (bold letters in the primer sequences). The resulting mutants were designated ST8SiaIIΔ1 to Δ6 and ST8SiaIVΔ1 to Δ5. The numbers refer to the location of the deleted N-glycosylation site as shown in Fig. 1 A. Open table in a new tab N-Glycosylation sites of ST8SiaII and ST8SiaIV were deleted by site-directed mutagenesis in a PCR approach using the mutation primers given in this table. The codon used for asparagine in the Asn-Xxx-Ser/Thr motif was changed to glutamine by the indicated nucleotide exchanges (bold letters in the primer sequences). The resulting mutants were designated ST8SiaIIΔ1 to Δ6 and ST8SiaIVΔ1 to Δ5. The numbers refer to the location of the deleted N-glycosylation site as shown in Fig. 1 A. CHO cells of the complementation group 2A10 are PSA-negative because of a defect in the ST8SiaIV gene and were generated by chemical mutagenesis of CHO K1 cells (36Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar). The cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 1:1 (Seromed) supplemented with 5% fetal calf serum and 1 mmsodium pyruvate and maintained in a humidified 5% CO2atmosphere at 37 °C. For transient transfections 2 × 106 cells were grown overnight on 100-mm tissue culture dishes, rinsed twice with PBS (10 mm sodium phosphate, pH 7.4/150 mm NaCl), and transfected with 4 μg of plasmid DNA and 24 μl of LipofectAMINE (Life Technologies, Inc.) in 4 ml of OptiMEM (Life Technologies, Inc.). After 7 h the transfection was stopped by adding 5 ml of medium containing 10% fetal calf serum. 24 h after the start of transfection the medium was substituted by 8 ml of medium containing 5% fetal calf serum, and the cells were harvested 48 h later. Alternatively, the cell supernatant was collected and used for measuring polysialyltransferase activityin vitro. For the selection of stable transfectants, cells were transfected as described and cultured 72 h later in the presence of 750 μg/ml G418 (Calbiochem). Colonies were picked and cloned by limiting dilution. Transfected cells of one 100-mm dish were lysed in 800 μl of ice-cold lysis buffer containing 50 mm Tris-HCl, pH 8.0, 1 mm MnCl2, 1% Nonidet P-40, 200 units of aprotinin, and 1 mmphenylmethylsulfonyl fluoride. Using the bicinchoninic acid (BCA) protein assay reagent (Pierce), the protein concentration was determined, and lysates were diluted with lysis buffer to a final concentration of 2.5 mg of protein/ml. For the immunoprecipitations 10 μl of wet Protein A-Sepharose covalently coupled with anti-HA mAb 12CA5 were added to 150 μl of lysate (375 μg of protein) and incubated overnight with gentle agitation at 4 °C. Thereafter, the beads were sedimented by centrifugation (5000 × g for 30 s) and washed twice with 50 mm Tris-HCl, pH 8.0, containing 0.5% Nonidet P-40. The washing buffer was carefully removed, and 15 μl of Laemmli sample buffer were added. Flag-HA-tagged ST8SiaII and -IV were transiently expressed in CHO-2A10 cells and immunoprecipitated with anti-HA mAb 12CA5 as described above but using 400 μl of lysate (1 μg of protein) for each immunoprecipitation. Beads were washed twice with TE buffer (10 mm Tris-HCl, pH 8.0/1 mm EDTA), and digests were performed in a final volume of 50 μl of TE buffer at 37 °C in a thermomixer (Eppendorf) at 1300 rpm for the indicated time points. Digests were started by adding 0.2 units PNGaseF for time points up to 15 min and 2 units for 1- and 3-h time points. Round-bottom microtiter plates (Greiner) were coated overnight with 0.2 μg of murine IgG (Pierce) at 4 °C and blocked with bovine serum albumin (1% in PBS) for 2 h at room temperature. After two washing steps with PBS, 10 μl of cell supernatant containing recombinant Protein A-fusion proteins were adsorbed for 1 h at 37 °C. In parallel, serial dilutions (4–0.2 ng/ml) of the recombinant IgG-binding fragment of Protein A (Sigma) were adsorbed to generate a standard curve. After the adsorption step, plates were washed three times with PBS, and 10 μl of 1: 50,000 diluted biotinylated anti-Protein A antibody SPA-27 were added per well. The plates were incubated for 1 h at 37 °C and washed three times with PBS. The bound biotinylated antibody was detected with streptavidin-horseradish peroxidase conjugate (Roche) using 10 μl of a 1:2000 dilution per well. After 30 min at 37 °C plates were washed three times with PBS and developed with 50 μl of substrate solution containing 5 μg of 3,3′,5,5′-tetramethylbenzidine, 100 mm sodium acetate, 100 mm citric acid, and 0.0045% H2O2. Reactions were stopped after 20 min by adding 25 μl of 2 n H2SO4, and the optical density was measured in an enzyme-linked immunosorbent assay reader at 450 nm. All samples were measured in serial dilutions, and triple values were analyzed for each dilution. 500 ng of wild-type and mutant Protein A-polysialyltransferases were adsorbed to 10 μl of IgG-Sepharose (Amersham Pharmacia Biotech). Beads were washed twice with 1 ml of PBS and were used either directly for measuring autopolysialylation or for measuring NCAM polysialylation after the adsorption of 1 μg of Protein A-NCAM. Enzyme-coupled beads were washed three times with 1 ml of reaction buffer containing 10 mm sodium cacodylate, pH 6.0, and 10 mmMnCl2, and assays were started by adding 125 nCi of CMP-[14C]Neu5Ac in 40 μl of reaction buffer to give a final volume of 50 μl. Incubations were performed in a thermomixer (Eppendorf) at 1300 rpm for 2 h at 37 °C and stopped by washing twice with 1 ml of PBS. One of two parallel samples was treated with 100 ng of endoNE in a final volume of 50 μl of PBS for 30 min at 37 °C. After two additional washing steps with 1 ml of PBS, residual buffer was removed, and 15 μl of Laemmli sample buffer were added. Samples were analyzed by SDS-PAGE and autoradiography. SDS-PAGE was performed according to Laemmli (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). Samples were subjected to gel electrophoresis under reducing conditions using 2.5% (v/v) β-mercaptoethanol. All samples were heated at 65 °C for 20 min, and 15 μl/slot were loaded on 10 or 7% polyacrylamide gels. For Western blot analysis, proteins were blotted onto nitrocellulose membranes (Schleicher & Schüell), and blots were developed as described (26Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. EMBO J. 1996; 15: 6943-6950Crossref PubMed Scopus (96) Google Scholar) with primary antibodies used in a concentration of 5 μg/ml. For autoradiographic analysis, gels were vacuum-dried immediately after electrophoresis and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech). LM-TK− cells were maintained in Dulbecco's modified Eagle's medium (Seromed) supplemented with 10% fetal calf serum. Stable cell lines expressing Flag-HA-tagged polysialyltransferases were generated as described for CHO-2A10 cells. Transient transfections were performed using the Effectene transfection kit (Qiagen). 48 h before transfection, 8 × 104cells were seeded per well of a 6-well plate containing four glass coverslips. The cells were washed once with PBS, and 1.6 ml of fresh medium were added. 0.8 μg of plasmid DNA of wild-type or mutant Flag-HA-ST8SiaII/IV constructs in 100 μl of EC buffer, 6.4 μl of Enhancer, 8 μl of Effectene, and 600 μl of medium were mixed and added to the cells. 24 h after transfection, the cells were washed with PBS, fixed in 4% paraformaldehyde for 20 min, and washed twice with PBS. For detection of Golgi-localized antigens cells were permeabilized with 0.2% Triton X-100 for 10 min at room temperature. For intracellular PSA staining, coverslips were incubated with endoNE (60 ng/ml in PBS) for 30 min at 37 °C to remove PSA on the cell surface prior to permeabilization. Immunodetections were carried out for 1 h at 37 °C with anti-PSA mAb 735 (5 μg/ml), anti-Flag mAb M5 (3.5 μg/ml), and rabbit anti-α-mannosidase II antiserum (1:2000) in 20% horse serum in PBS. After washing four times with PBS, the cells were incubated for 30 min at 37 °C with sheep anti-mouse IgG-Cy3 (1:300, Sigma) and goat anti-rabbit IgG-ALEXA 488 (1:500, Molecular Probes) in 20% horse serum in PBS. The cells were washed three times with PBS, and coverslips were mounted in Moviol and analyzed under a Zeiss Axiophot fluorescence microscope. The schematic representations of polysialyltransferases shown in Fig.1 A illustrate the number and relative locations of the predicted five and six N-glycan attachment sites in ST8SiaIV (36Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar) and ST8SiaII (38Livingston B.D. Paulson J.C. J. Biol. Chem. 1993; 268: 11504-11507Abstract Full Text PDF PubMed Google Scholar), respectively. To determine how many of these sites are effectively used in the native proteins, full-length murine ST8SiaII and hamster ST8SiaIV, N-terminally tagged with Flag and HA epitopes, were analyzed by limited PNGaseF digestions followed by SDS-PAGE and Western blot analysis (Fig.1, B and C). Native Flag-HA-ST8SiaIV migrated as a focused band of 55 kDa (Fig. 1 B, lane 1). After 180 min PNGaseF treatment the completely deglycosylated protein migrated with the expected molecular mass of 44 kDa (calculated 44.1 kDa). Partially deglycosylated enzymes were displayed after 2, 5, 15, and 60 min. In agreement with the approximate molecular mass of a single N-linked oligosaccharide chain, individual bands were separated by about 3 kDa. A total of six bands was displayed, demonstrating that five N-linked oligosaccharides are attached to ST8SiaIV. Fig. 1 C shows the results obtained for Flag-HA-ST8SiaII. The native protein migrated as a double band at 60 and 57 kDa (lane 1), suggesting the presence of two glycosylation variants. Complete deglycosylation (lane 6) resulted in a protein of 45 kDa (calculated 45.3 kDa), and partial digestions (see lanes 2–5) displayed a total of seven bands, indicating the presence of six N-linked carbohydrate chains in ST8SiaII. In summary these data show that all predicted N-glycan attachment sites are used in the recombinant polysialyltransferases isolated from CHO cells. For convenience, the numbers as shown in Fig.1 A will be used in the following text to refer to individualN-glycan attachment sites. To estimate the importance of individual carbohydrate chains for the enzymatic activity of the polysialyltransferases, N-glycosylation sites were inactivated, individually or in different combinations, by replacing asparagine in the glycosylation sequon Asn-XXX-Ser/Thr with glutamine (see Table I for details). 17 mutants were generated for ST8SiaIV (Fig.2) and 29 for ST8SiaII (Fig.3) including aglyco-variants (∅) and mutants containing only single N-glycan attachment sites (series +1, +2, etc.). Activities of mutant proteins were tested in vivo and in vitro. In vivo activities were measured by complementation analysis in CHO-2A10 cells. In CHO wild-type cells, ST8SiaIV is responsible for PSA expression. Because of a defect in the ST8SiaIV gene, cells of the complementation group 2A10 are unable to polysialylate NCAM (18Windfuhr M" @default.
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• W2068746509 title "The Impact of N-Glycosylation on the Functions of Polysialyltransferases" @default.
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