Differential and Cooperative Polysialylation of the Neural Cell Adhesion Molecule by Two Polysialyltransferases, PST and STX
1998; Elsevier BV; Volume: 273; Issue: 43 Linguagem: Inglês
10.1074/jbc.273.43.28524
ISSN1083-351X
AutoresKiyohiko Angata, Misa Suzuki, Minoru Fukuda,
Tópico(s)Ubiquitin and proteasome pathways
ResumoPST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (NCAM), and these two polysialyltransferases often exist together in the same tissues. To determine the individual and combined roles of PST and STX in polysialic acid synthesis, in the present study we asked if PST and STX differ in the acceptor requirement and if PST and STX act together in polysialylation of NCAM. We first examined whether PST and STX differ in the requirement of sialic acid and core structures of N-glycans attached to NCAM. Polysialic acid was formed well on Lec4 and Lec13 cells, which are defective in N-acetylglucosaminyltransferase V and GDP-fucose synthesis, respectively, demonstrating that a side chain elongating from GlcNAcβ1→6Manα1→6R and α-1,6-linked fucose are not required. PST and STX were found to add polysialic acid on NCAM·Fc molecules sialylated by α-2,3- or α-2,6-linkage in vitro, but not on NCAM·Fc lacking either sialic acid. These results indicate that both PST and STX have relatively broad specificity onN-glycan core structures in NCAM and no remarkable difference exists between PST and STX for the requirement of core structures and sialic acid attached to the N-glycans of NCAM. We then, using various N-glycosylation site mutants of NCAM, discovered that PST strongly prefer the sixthN-glycosylation site, which is the closest to the transmembrane domain, over the fifth site. STX slightly prefer the sixth N-glycosylation site over the fifthN-glycosylation site. The results also demonstrated that polysialic acid synthesized by PST is larger than that synthesized by STX in vitro. Moreover, a mixture of PST and STX more efficiently synthesized polysialic acid on NCAM than PST or STX alone. These results suggest that polysialylation of NCAM is influenced by the difference between PST and STX in their preference forN-glycosylation sites on NCAM. The results also suggest that PST and STX form polysialylated NCAM in a synergistic manner. PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (NCAM), and these two polysialyltransferases often exist together in the same tissues. To determine the individual and combined roles of PST and STX in polysialic acid synthesis, in the present study we asked if PST and STX differ in the acceptor requirement and if PST and STX act together in polysialylation of NCAM. We first examined whether PST and STX differ in the requirement of sialic acid and core structures of N-glycans attached to NCAM. Polysialic acid was formed well on Lec4 and Lec13 cells, which are defective in N-acetylglucosaminyltransferase V and GDP-fucose synthesis, respectively, demonstrating that a side chain elongating from GlcNAcβ1→6Manα1→6R and α-1,6-linked fucose are not required. PST and STX were found to add polysialic acid on NCAM·Fc molecules sialylated by α-2,3- or α-2,6-linkage in vitro, but not on NCAM·Fc lacking either sialic acid. These results indicate that both PST and STX have relatively broad specificity onN-glycan core structures in NCAM and no remarkable difference exists between PST and STX for the requirement of core structures and sialic acid attached to the N-glycans of NCAM. We then, using various N-glycosylation site mutants of NCAM, discovered that PST strongly prefer the sixthN-glycosylation site, which is the closest to the transmembrane domain, over the fifth site. STX slightly prefer the sixth N-glycosylation site over the fifthN-glycosylation site. The results also demonstrated that polysialic acid synthesized by PST is larger than that synthesized by STX in vitro. Moreover, a mixture of PST and STX more efficiently synthesized polysialic acid on NCAM than PST or STX alone. These results suggest that polysialylation of NCAM is influenced by the difference between PST and STX in their preference forN-glycosylation sites on NCAM. The results also suggest that PST and STX form polysialylated NCAM in a synergistic manner. the neural cell adhesion molecule endoneuraminidase polysialyltransferase (ST8Sia IV) sialyltransferase X (ST8Sia II) the hinge and constant region of human IgG immunoglobulin-like fluorescein isothiocyanate high performance liquid chromatography Chinese hamster ovary. Polysialic acid is a developmentally regulated carbohydrate composed of a linear homopolymer of α-2,8-linked sialic acid (1Finne J. J. Biol. Chem. 1982; 257: 11966-11970Abstract Full Text PDF PubMed Google Scholar). NCAM1 is highly polysialylated in embryonic tissues. In contrast, the majority of NCAM in adult tissues lacks this unique glycan, but polysialylated NCAM is present in the olfactory bulb and hippocampus of adult brain, where neural regeneration persists (2Edelman G.M. Annu. Rev. Biochem. 1985; 54: 135-169Crossref PubMed Scopus (276) Google Scholar, 3Rutishauser U. Landmesser L. Trends Neurosci. 1996; 19: 422-427Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). There is increasing evidence that polysialylated NCAM promotes cell migration and enhances neurite outgrowth and branching during development and neural regeneration (2Edelman G.M. Annu. Rev. Biochem. 1985; 54: 135-169Crossref PubMed Scopus (276) Google Scholar, 3Rutishauser U. Landmesser L. Trends Neurosci. 1996; 19: 422-427Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 4Kiss J.-Z. Rougon G. Curr. Opin. Neurobiol. 1997; 7: 640-646Crossref PubMed Scopus (206) Google Scholar). Polysialic acid is thought to modulate the functional properties of NCAM by rendering it less adhesive to itself (homophilic binding) (5Hoffman S. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5762-5766Crossref PubMed Scopus (515) Google Scholar, 6Sadoul R. Hirn M. Deagostini-Bazin H. Rougon G. Goridis C. Nature. 1983; 304: 347-349Crossref PubMed Scopus (291) Google Scholar) or to other cell surface molecules (heterophilic binding). In the latter case, it has been shown that NCAM engages in interactions with L1 on the same membrane (cis-interaction) (7Kadmon G. Kowitz A. Altevogt P. Schachner M. J. Cell Biol. 1990; 110: 193-208Crossref PubMed Scopus (244) Google Scholar). The studies on NCAM knockout mice demonstrated an abnormal formation in the olfactory bulb and hippocampus and a defect in spatial learning and memory (8Cremer H. Lange R. Christoph A. Plomann M. Vopper G. Roes J. Brown R. Baldwin S. Kreamer P. Scheff S. Bartheis D. Rajewsky K. Wille W. Nature. 1994; 367: 455-459Crossref PubMed Scopus (899) Google Scholar, 9Tomasiewicz H. Ono K. Yee D. Thompson C. Goridis C. Rutishauser U. Magnuson T. Neuron. 1993; 11: 1163-1174Abstract Full Text PDF PubMed Scopus (436) Google Scholar). By using NCAM knockout mice and endoneuraminidase (endo-N) treatment, recent studies have demonstrated that polysialic acid is required for the migration of cells in the subventricular zone of the olfactory bulb (10Hu H. Tomasiewicz H. Magnuson T. Rutishauser U. Neuron. 1996; 16: 735-743Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Similarly, endo-N treatment of hippocampal cells in organotypic slice cultures was shown to prevent the induction of long term potentiation, presumably by impairing the induction of synaptic plasticity (11Muller D. Wang C. Skibo G. Toni N. Cremer H. Calaora V. Rougon G. Kiss J.Z. Neuron. 1996; 17: 413-422Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). These results, taken together, strongly suggest that polysialylated NCAM plays a critical role during development and neural regeneration. The cDNAs encoding polysialyltransferases have been cloned, and these enzymes are called PST and STX (12Livingston B.D. Paulson J.C. J. Biol. Chem. 1993; 268: 11504-11507Abstract Full Text PDF PubMed Google Scholar, 13Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 14Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 15Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Kojima N. Yoshida Y. Kurosawa N. Lee Y.C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar, 17Yoshida Y. Kojima N. Tsuji S. J. Biochem. (Tokyo). 1995; 118: 658-664Crossref PubMed Scopus (79) Google Scholar). PST (also called ST8Sia IV) and STX (also called ST8Sia II) belong to a member of the sialyltransferase gene family which shares the two conserved amino acid sequences, sialyl motif L and S (18Datta A.K. Paulson J.C. J. Biol. Chem. 1995; 270: 1497-1500Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). PST and STX are highly homologous to each other and have 59% identity at the amino acid level (13Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 14Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 15Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Kojima N. Yoshida Y. Kurosawa N. Lee Y.C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar, 17Yoshida Y. Kojima N. Tsuji S. J. Biochem. (Tokyo). 1995; 118: 658-664Crossref PubMed Scopus (79) Google Scholar). Consistent with the presumed roles of polysialic acid, it has been shown that polysialic acid synthesized by PST or STX facilitates neurite outgrowth (14Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). After transfecting HeLa cells with human PST or STX and NCAM cDNAs or NCAM cDNA alone, they were used as the substratum for neurite outgrowth assay. When neurons derived from embryonic chick dorsal root ganglia were grown on these substrata, neurites were much longer and more branched on the substratum cells expressing polysialic acid and NCAM than those on the substratum expressing NCAM alone (14Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). By using an in vitro assay system, both PST and STX were shown to add polysialic acid to fetuin and soluble chimeric NCAM (20Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21Kojima N. Tachida Y. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 22Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. Curr. Biol. 1996; 6: 1188-1191Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This demonstrates that either PST or STX alone can form polysialic acid by adding the first α-2,8-linked sialic acid to α-2,3-linked sialic acid in an acceptor, followed by the multiple addition of α-2,8-linked sialic acid residues. In this context, PST and STX thus appear to share common enzymatic properties. In contrast to this, it has been suggested that the synthesis of polysialic acid attached to α-2,6-linked sialic acid in mucin-typeO-glycans requires an "initiase" α-2,8-sialyltransferase (23Kitazume S. Kitajima K. Inoue S. Inoue Y. Troy F.A., II J. Biol. Chem. 1994; 269: 10330-10340Abstract Full Text PDF PubMed Google Scholar). Multiple residues of α-2,8-linked sialic acid are added by an "elongase" α-2,8-sialyltransferase possibly on a preformed α-2,8-linked sialic acid. However, this conclusion was mainly based on the structural determination of poly- and oligosialic acid present in different stages of trout egg development and the initiase was not assayed (23Kitazume S. Kitajima K. Inoue S. Inoue Y. Troy F.A., II J. Biol. Chem. 1994; 269: 10330-10340Abstract Full Text PDF PubMed Google Scholar). Kojima et al. (21Kojima N. Tachida Y. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), on the other hand, reported that PST and STX require α-1,6-linked fucose attached to N-glycans of NCAM and that PST forms polysialic acid on all forms of NCAM while STX does not form polysialic acid on NCAM-120, suggesting a difference in the acceptor specificity for two polysialyltransferases (24Kojima N. Tachida Y. Tsuji S. J. Biochem. (Tokyo). 1997; 122: 1265-1273Crossref PubMed Scopus (32) Google Scholar). Recent studies indicate that both PST and STX are highly expressed in early stages of mouse or rat development and that this amount is at a maximum before birth (25Kurosawa N. Yoshida Y. Kojima N. Tsuji S. J. Neurochem. 1997; 69: 494-503Crossref PubMed Scopus (61) Google Scholar, 26Phillips G.R. Krushel L.A. Crossin K.L. Dev. Brain Res. 1997; 102: 143-155Crossref PubMed Scopus (52) Google Scholar, 27Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Crossref PubMed Scopus (122) Google Scholar, 28Wood G.K. Liang J.J. Flores G. Ahmad S. Quirion R. Srivastava L.K. Mol. Brain Res. 1997; 51: 69-81Crossref PubMed Scopus (30) Google Scholar). In mouse, 10 days after birth, STX is dramatically decreased while PST is moderately decreased during development (25Kurosawa N. Yoshida Y. Kojima N. Tsuji S. J. Neurochem. 1997; 69: 494-503Crossref PubMed Scopus (61) Google Scholar, 27Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Crossref PubMed Scopus (122) Google Scholar). On the other hand, both PST and STX are present in many of the cells so far examined. For example, polysialic acid in the hippocampus in adult brain appears to be synthesized by concerted actions of PST and STX (19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 26Phillips G.R. Krushel L.A. Crossin K.L. Dev. Brain Res. 1997; 102: 143-155Crossref PubMed Scopus (52) Google Scholar, 28Wood G.K. Liang J.J. Flores G. Ahmad S. Quirion R. Srivastava L.K. Mol. Brain Res. 1997; 51: 69-81Crossref PubMed Scopus (30) Google Scholar). These results combined together indicate that PST and STX may individually have distinct functions, but it is not clear why two similar enzymes are expressed in common regions of the brain. These results prompted us to examine how PST and STX differ in the polysialylation of NCAM, and how two enzymes act together on NCAM. In the present study, we first examined the requirement in the core structure and sialic acid residue attached to N-glycans for polysialylation by PST and STX. We found that α-1,6-linked fucose or the side chain of Galβ1→4GlcNAcβ1→6Manα1→6Manα1→R is not necessary for the synthesis of polysialic acid, but α-2,3- or α-2,6-linked sialic acid must be present. We then found that PST and STX differ in polysialylation at different N-glycosylation sites of NCAM. Finally, we demonstrated that the actions of PST and STX on NCAM are not mutually exclusive, but instead cooperative. Human PST cDNA encoding the full-length PST in pcDNAI, pcDNAI-PST and pcDNAI-A·PST harboring cDNA encoding a soluble PST chimeric with protein A was constructed as described previously (14Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 20Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). pcDNAI-STX harboring the full-length cDNA of human STX was cloned as described previously (19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). pIG-NCAM·Fc encoding a soluble NCAM fused with Fc portion of human IgG in pIG vector (29Simmons D.L. Hartly D. Cellular Interactions in Development. Oxford University Press, Oxford1993: 93-127Google Scholar) was kindly provided by Dr. David Simmons, Oxford University. pIG vector was modified from pCDM8. HeLa cells were stably transfected with pIG-NCAM·Fc, pSV2neo, pcDNAI-PST or pcDNAI-STX as described (19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). After selection with G418, HeLa cells expressing both polysialic acid and NCAM·Fc were established using immunostaining with anti-polysialic acid antibody, 12F8 (Ref. 30Bourne S.P. Patel K. Walsh F. Popham C.J. Coakham H.B. Kemshead J.T. J. Neuro-Oncol. 1991; 10: 111-119Crossref PubMed Scopus (48) Google Scholar; PharMingen), or anti-NCAM antibody, CD56 (Becton Dickinson) on permeabilized cells. Similarly, HeLa cells were transfected with pIG-NCAM·Fc and pSV2neo, and those cells expressing NCAM·Fc, HeLa-NCAM·Fc, were selected. The permeabilization of cell membranes by saponin and immunostaining protocol was as described previously (31Williams M.A. Fukuda M. J. Cell Biol. 1990; 111: 955-966Crossref PubMed Scopus (192) Google Scholar). The selected cell lines were metabolically labeled with [3H]glucosamine for 24 h as described previously (32Lee N. Wang W.-C. Fukuda M. J. Biol. Chem. 1990; 265: 20476-20487Abstract Full Text PDF PubMed Google Scholar). NCAM·Fc bound to protein A-agarose (Pierce) was first suspended in 10 mm Tris-HCl buffer, pH 8.0, containing 0.1% SDS and 50 mm 2-mercaptoethanol. After incubation for 10 min at 37 °C, 0.75% Nonidet P-40 (the final concentration) was added to the sample, and the sample was incubated withN-glycanase (Boehringer Mannheim) (0.8 units/100 μl of beads) with rotation for 24 h at 37 °C. The releasedN-glycans were recovered in the supernatant after centrifugation. In order to remove N-glycanase, the supernatant was mixed with 50% (final concentration) of phenol, and the water phase after centrifugation was washed once with chloroform to remove a trace amount of phenol. The sample in the water phase was then applied to a column (1.0 × 110 cm) of Sephadex G-50 (superfine, Amersham Pharmacia Biotech) equilibrated with 0.1 mNH4HCO3. The column was eluted at 6 ml/h and fractions (1.4 ml/fraction) were collected. Aliquots of each fraction were taken for the determination of radioactivity and those fractions containing N-glycans were pooled, lyophilized, and desalted by Sephadex G-25 (Amersham Pharmacia Biotech) gel filtration eluted with water. The desaltedN-glycans were sequentially digested with NANase II and NANase III (Glyko). NANase II cleaves α-2,3-linked and α-2,6-linked sialic acids. NANase III cleaves α-2,8-linked sialic acid in addition to α-2,3- and α-2,6-linked sialic acids. After each digestion, the sample was subjected to Sephadex G-50 gel filtration for fractionation then Sephadex G-25 gel filtration to remove salts, as described above. Wild-type CHO, CHO mutant Lec2 (33Deutscher S.L. Nuwayhid N. Stanley P. Barak Briles E.I. Levine A.Y.A. Ben-Ahser E. Aloni Y. Razin A. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (195) Google Scholar), Lec4 (34Stanley P. Vivona G. Atkinson P.H. Arch. Biochem. Biophys. 1984; 230: 363-374Crossref PubMed Scopus (15) Google Scholar), and Lec13 (35Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (68) Google Scholar) cells were transiently transfected with pcDNAI-PST or pcDNAI-STX using LipofectAMINE (Life Technologies, Inc.) as described (14Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar). Lec2, Lec4, and Lec13 cells were shown to be defective in CMP-sialic acid transporter,N-acetylglucosaminyltransferase V, and GDP-d-mannose-4,6-dehydratase, respectively (33Deutscher S.L. Nuwayhid N. Stanley P. Barak Briles E.I. Levine A.Y.A. Ben-Ahser E. Aloni Y. Razin A. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 34Stanley P. Vivona G. Atkinson P.H. Arch. Biochem. Biophys. 1984; 230: 363-374Crossref PubMed Scopus (15) Google Scholar, 35Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (68) Google Scholar). Wild-type CHO and Lec2 cells were obtained from American Type Culture Collection while Lec4 and Lec13 cells were kindly provided by Dr. Pamela Stanley, Albert Einstein College of Medicine, New York, NY. Forty-eight h after the transfection, the cells were dispersed into single cells by the cell dissociation solution (Cell & Molecular Technologies, Lavallete, NJ), and then incubated with anti-polysialic acid antibody (12F8) followed by FITC-conjugated goat affinity-purified (Fab′)2 fragment specific to rat IgM. The cells were then analyzed by flow cytometry as described (36Tsuboi S. Fukuda M. EMBO J. 1997; 16: 6364-6373Crossref PubMed Scopus (83) Google Scholar). First, pcDNAI-A, which encodes the signal peptide of human colony-stimulating factor and a IgG binding domain of Staphylococcus aureus protein A, was prepared as follows. pAMoA-GD3 (37Sasaki K. Kurata K. Kojima N. Kurosawa N. Ohta S. Hanai N. Tsuji S. Nishi T. J. Biol. Chem. 1994; 269: 15950-15956Abstract Full Text PDF PubMed Google Scholar) was digested by SalI and BamHI, and cDNA encoding the signal peptide-protein A chimera was cloned into XhoI and BamHI sites of pBluescript II (Stratagene). This cDNA excised by KpnI and BamHI was cloned into pcDNA3 (Invitrogen). ByBamHI digestion and partial HindIII digestion of this plasmid, cDNA was excised and cloned into HindIII and BamHI sites of pcDNAI. This vector, pcDNAI-A, is a universal vector to clone a catalytic domain of glycosyltransferases using BamHI site and 3′-restriction sites such asEcoRI, EcoRV, NotI, XhoI, and XbaI. STX cDNA was cloned from the human fetal brain mRNAs using Superscript™ II (Life Technologies, Inc.) and the 3′-primer, ASTX-3–1, as described previously (19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Using the cDNA as a template, the sequence between nucleotides 96 and 1128 was amplified. Upstream and downstream primers used for this polymerase chain reaction were 5′GGAAGATCTCGGGAATTCGGGAGGCAG-3′ (BglII site shown by underline) and 5′-CCGCTCGAGCTACGTGCCCCATCGCACTG-3′ (XhoI site shown by underline), respectively. This polymerase chain reaction product encoding the catalytic domain of STX was digested byBglII and XhoI and cloned into theBamHI and XhoI sites of pcDNAI-A described above, resulting in pcDNAI-A·STX. Targeted mutations of asparagine residues within the fifth immunoglobulin domain of NCAM were carried out in double-stranded DNA as described (38Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1078) Google Scholar), using the Chameleon™ double-stranded site-directed mutagenesis kit (Stratagene). The cDNA encoding the fifth Ig domain of human NCAM was excised between ApaI (nucleotide 1227, nucleotide 1 encodes the initiation methionine codon) and EcoRI (nucleotide 1471) sites. This cDNA fragment was cloned into pBluescript II and used as a template for site-directed mutagenesis. Three oligonucleotides were synthesized according to the nucleotide sequence of NCAM (39Dickson G. Gower H.J. Barton C.H. Prentice H.M. Elsom V.L. Moore S.E. Cox R.D. Quinn C. Putt W. Walsh F.S. Cell. 1987; 50: 1119-1130Abstract Full Text PDF PubMed Scopus (167) Google Scholar) for site-directed mutagenesis. For example, 5′-TGGGAGGGGAACCAGGTGCAGATCACCTGCGAGGTATTTG-3′ (nucleotides 1249–1288) was used for the mutation from Asn-423 to Gln-423. For the mutation of Asn-449 and Asn-478, primers corresponding to nucleotides 1326–1365 and nucleotides 1417–1449 were used, where the Asn codon was mutated to Gln codon (CAA and CAG, respectively). First, one of those oligonucleotides and the ScaI → MluI primer, provided by the supplier, were annealed and the DNA was extended and ligated. The resultant plasmids were then digested byScaI to remove the template DNA, and then amplified in XLmutS competent cells. Plasmids recovered were digested with ScaI again, removing template double-stranded plasmids. After transformation into XL1-Blue competent cells, each colony was isolated to confirm the sequence. The ApaI-EcoRI fragment of the cDNA with the mutated sequence was then put back into pIG-NCAM·Fc, replacing the wild-type corresponding sequence. In order to obtain cDNA with double mutations, the above procedure was repeated using the mutated cDNA as the template with another oligonucleotide and the MluI → ScaI oligonucleotide primer, provided by Stratagene. After the sequential third mutation, cDNA containing mutations at all of the threeN-glycosylation sites in the fifth immunoglobulin domain were obtained. All constructs were verified by nucleotide sequencing performed as described (19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Once HeLa-NCAM·Fc cells reached 70% confluence, the medium was replaced with serum-free medium, Opti-MEM I (Life Technologies, Inc.) and cultured for 24 h. NCAM·Fc in the spent medium was adsorbed to protein A-agarose, and was eluted from the resin by a gentle Ag/Ab elution buffer (Pierce) (36Tsuboi S. Fukuda M. EMBO J. 1997; 16: 6364-6373Crossref PubMed Scopus (83) Google Scholar). The eluted material was desalted by repeated concentration and addition of 20 mm Tris-HCl, pH 7.4 containing 0.05% Tween 20 using Microcon 30 (Amicon). Similarly, COS-1 cells were transiently transfected with pIG-NCAM·Fc or pIG harboring mutated NCAM·Fc using LipofectAMINE as described above. NCAM·Fc was purified in the same procedure as described above. The amount of NCAM·Fc was measured using BCA protein assay (Pierce) and bovine serum albumin as a standard. The concentration of each mutated NCAM·Fc was adjusted after measuring the amount by Western blotting using peroxidase-conjugated goat IgG specific to the Fc protein of human IgG (Cappel) and ECL kit (Amersham Pharmacia Biotech). pcDNAI-A·PST and pcDNAI-A·STX were separately transfected into HeLa cells with pSV2neo using LipofectAMINE as described (19Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). After selection with G418, clonal cell lines expressing these chimeric proteins were selected following permeabilization of cells and staining with FITC-conjugated human IgG, which reacts with the protein A portion of the chimeric protein. The clonal cell lines were cultured in macrophage-SFM medium (Life Technologies, Inc.) for 24 h and the chimeric enzymes secreted into the culture medium was adsorbed to IgG-Sepharose 6FF (Amersham Pharmacia Biotech) after titrating the pH of the spent medium to 8.0. The resin was collected by centrifugation and washed as described (20Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,21Kojima N. Tachida Y. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) except the following modification. The resin was finally suspended in an equal volume of a serum free medium of macrophage-SFM and used as the enzyme solution. The enzyme activity was measured as described previously (20Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21Kojima N. Tachida Y. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The substrate solution contained 2 μg of NCAM·Fc dissolved in 50 μl of 0.1 m sodium cacodylate buffer, pH 6.0, containing 5 mm MnCl2 and 2 mmCaCl2, 1% Triton CF-54, 2.4 nmol (0.7 μCi) of CMP-[14C]NeuNAc. To this substrate solution, 50 μl of the enzyme solution prepared above was added and the reaction mixture was incubated at 37 °C for various times. At the end of incubation, the reaction mixture was centrifuged and the supernatant was recovered. To 20 μl of this supernatant, 20 μl of the sample buffer for SDS-polyacrylamide gel electrophoresis (2× concentration) was added, heated, and subjected to electrophoresis followed by fluorography using the same conditions as described (20Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The amount of incorporated [14C]NeuNAc into NCAM was measured on each fluorograph by NIH Image version 1.61. The product, NCAM·Fc, was digested with Pronase (Calbiochem) in 400 μl of 0.1 m Tris-HCl, pH 8.0 containing 1 mmCaCl2 as described (32Lee N. Wang W.-C. Fukuda M. J. Biol. Chem. 1990; 265: 20476-20487Abstract Full Text PDF PubMed Google Scholar). The digestion was stopped by phenol extraction and glycopeptides in the aqueous phase, after washing with chloroform, were fractionated by Mono-Q HPLC using a procedure modified from the one reported (40Livingston B.D. Jacobs J.L. Glick M.C. Troy F.A., II J. Biol. Chem. 1988; 263: 9443-9448Abstract Full Text PDF PubMed Google Schola
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