Polysialic Acid and Mucin Type O-Glycans on the Neural Cell Adhesion Molecule Differentially Regulate Myoblast Fusion
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m308316200
ISSN1083-351X
AutoresMisa Suzuki, Kiyohiko Angata, Jun Nakayama, Minoru Fukuda,
Tópico(s)Signaling Pathways in Disease
ResumoPolysialic acid attached to the neural cell adhesion molecule (NCAM) is thought to play a critical role in development. NCAM in muscle tissue contains a muscle-specific domain (MSD) to which mucin type O-glycans are attached. In the present study, using the C2C12 myoblast system, we show that NCAM containing MSD is increasingly expressed on the cell surface as myotubes form. Polysialic acid is primarily attached to N-glycans of NCAM, and polysialylated NCAM is expressed on the outer surface of myotube bundles. By transfecting cDNAs encoding wild type and mutant forms of NCAM, we found that NCAM containing MSD facilitates myoblast fusion, and this effect is diminished by mutating O-glycosylation sites at MSD. By contrast, forced expression of polysialic acid in early differentiation stages reduces myotube formation and delays the expression of NCAM containing the MSD domain. Strikingly, inhibition of polysialic acid synthesis by antisense DNA approach induced differentiation in both human rhabdomyosarcoma cells, which overexpress polysialic acid, and C2C12 cells. These results indicate that polysialic acid and mucin type O-glycans on NCAM differentially regulate myoblast fusion, playing critical roles in muscle development. Polysialic acid attached to the neural cell adhesion molecule (NCAM) is thought to play a critical role in development. NCAM in muscle tissue contains a muscle-specific domain (MSD) to which mucin type O-glycans are attached. In the present study, using the C2C12 myoblast system, we show that NCAM containing MSD is increasingly expressed on the cell surface as myotubes form. Polysialic acid is primarily attached to N-glycans of NCAM, and polysialylated NCAM is expressed on the outer surface of myotube bundles. By transfecting cDNAs encoding wild type and mutant forms of NCAM, we found that NCAM containing MSD facilitates myoblast fusion, and this effect is diminished by mutating O-glycosylation sites at MSD. By contrast, forced expression of polysialic acid in early differentiation stages reduces myotube formation and delays the expression of NCAM containing the MSD domain. Strikingly, inhibition of polysialic acid synthesis by antisense DNA approach induced differentiation in both human rhabdomyosarcoma cells, which overexpress polysialic acid, and C2C12 cells. These results indicate that polysialic acid and mucin type O-glycans on NCAM differentially regulate myoblast fusion, playing critical roles in muscle development. During mammalian embryonic development, muscle development is initiated once mesenchymal cells commit to become myoblasts. Myoblasts then align and fuse, forming multinucleated myotubes, and mature myotubes are bundled together, forming sarcolemma. In parallel, each myotube starts contraction, receives innervation from motor and sensory neurons, and eventually forms muscle tissue. In this process, one of the most intriguing steps is myoblast fusion. Myoblast fusion consists of multiple steps: expression of the fusion competent phenotype, specific myoblast-myoblast recognition and alignment, cell adhesion and electrical coupling, and fusion of lipid bilayer membrane (1.Knudsen K.A. Wilschut J. Hoekstral D. Membrane Fusion. Dekker, New York1991: 601-626Google Scholar). It has been reported that anti-NCAM 1The abbreviations used are: NCAMthe neural cell adhesion moleculeGPIglycosylphosphatidylinositolMSDmuscle-specific domainPNApeanut agglutininCFPcyan fluorescence proteinDMEDulbecco's modified Eagle's mediumFITCfluorescein isothiocyanatePBSphosphate-buffered salineTMtransmembraneFGFfibroblast growth factorRTreverse transcriptase.1The abbreviations used are: NCAMthe neural cell adhesion moleculeGPIglycosylphosphatidylinositolMSDmuscle-specific domainPNApeanut agglutininCFPcyan fluorescence proteinDMEDulbecco's modified Eagle's mediumFITCfluorescein isothiocyanatePBSphosphate-buffered salineTMtransmembraneFGFfibroblast growth factorRTreverse transcriptase. antibody inhibits aggregation of fusion-competent chick embryo myoblasts (2.Knudsen K.A. McElwee S.A. Smith L. Dev. Biol. 1990; 138: 159-168Crossref PubMed Scopus (93) Google Scholar), indicating that cell-cell adhesion via NCAM apparently initiates myoblast fusion. In addition, integrins, N-cadherin, VCAM, and VLA4 are thought to be involved (see Ref. 3.Schwander M. Leu M. Stumm M. Dorchies O.M. Ruegg S.T. Schittny J. Muller U. Dev. Cell. 2003; 4: 673-685Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar and references herein).NCAM is found in several isoforms due to alternative mRNA splicing of more than 19 exons. In mammalian brain, the glycosylphosphatidylinositol (GPI)-anchored 120-kDa isoform and transmembrane 140- and 180-kDa isoforms are primarily expressed. In muscles, three isoforms have been reported: a GPI-anchored 125-kDa isoform and transmembrane 140- and 155-kDa isoforms (4.Walsh F.S. Parekh R.B. Moore S.E. Dickson G. Barton C.H. Gower H.J. Dwek R.A. Rademacher T.W. Development. 1989; 105: 803-811PubMed Google Scholar). Muscle-specific domain (MSD) was identified in the cDNA of the 125-kDa NCAM isoform obtained from human skeletal muscle (5.Dickson 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). MSD is encoded by four different exons, MSD1a, MSD1b, MSD1c, and codon AAG. The full-length MSD sequence consists of 35 amino acids inserted between two fibronectin type III domains of NCAM. MSD insertion is observed only in skeletal muscle and heart and not in other tissues. O-Glycosylation of MSD was shown by peanut agglutinin (PNA) lectin affinity column chromatography and structural analysis (4.Walsh F.S. Parekh R.B. Moore S.E. Dickson G. Barton C.H. Gower H.J. Dwek R.A. Rademacher T.W. Development. 1989; 105: 803-811PubMed Google Scholar)Transfection studies using in vitro myoblast culture systems indicated that NCAM containing MSD promotes myoblast fusion. Dickson et al. transfected GPI-anchored human NCAM with (125 kDa) or without (120 kDa) MSD into C2 myoblast cells and found that 125-kDa NCAM enhanced myoblast fusion, whereas the 120-kDa isoform did not (6.Dickson G. Peck D. Moore S.E. Barton C.H. Walsh F.S. Nature. 1990; 344: 348-351Crossref PubMed Scopus (112) Google Scholar). Similarly, in transgenic mice overexpressing the human 125-kDa NCAM that contains MSD, secondary myotube formation is enhanced (7.Fazeli S. Wells D.J. Hobbs C. Walsh F.S. J. Cell Biol. 1996; 135: 241-251Crossref PubMed Scopus (27) Google Scholar). These results suggest that MSD motif promotes myoblast fusion. However, it is not known whether O-glycans attached to MSD or the amino acid sequence of MSD contribute to this activity.It has been reported that polysialic acid, another modification of NCAM, plays a critical role in neural development. Polysialic acid is a homopolymer of α2,8-linked sialic acid, which can be extended as many as 60 units in the developing nervous system. Polysialic acid is synthesized by two polysialyltransferases, ST8Sia II and ST8Sia IV (8.Angata K. Fukuda M. Biochimie (Paris). 2003; 85: 195-206Crossref PubMed Scopus (187) Google Scholar, 9.Livingston B.D. Paulson J.C. J. Biol. Chem. 1993; 268: 11504-11507Abstract Full Text PDF PubMed Google Scholar, 10.Eckhardt M. Muhlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 11.Nakayama 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, 12.Kojima N. Yoshida Y. Kurosawa N. Lee Y.C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar, 13.Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). NCAM is the most prominent acceptor molecule of polysialic acid, and the fifth and sixth N-glycosylation sites in the immunoglobulin-like domain 5 of NCAM are known to be preferentially polysialylated (14.Nelson R.W. Bates P.A. Rutishauser U. J. Biol. Chem. 1995; 270: 17171-17179Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15.Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16.Close B.E. Mendiratta S.S. Geiger K.M. Broom L.J. Ho L.L. Colley K.J. J. Biol. Chem. 2003; 278: 30796-30805Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Gene inactivation of ST8Sia IV is associated with impaired long term potentiation in Schaffer collateral-CA1 synapses of the adult hippocampus (17.Eckhardt 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). This defect is apparently due to a substantial decrease of polysialic acid in the CA1 region. These and other studies suggest that polysialic acid attenuates NCAM-NCAM homophilic interaction, allowing cells expressing polysialic acid to migrate and/or extend.In the present study, we employed the C2C12 mouse myoblast culture system to determine the roles of NCAM glycosylation in muscle development. C2C12 cells were derived from so-called satellite muscle cells and can be maintained as myoblast cells. When C2C12 cells are cultured in a low concentration of horse serum, they differentiate and form myotubes within 5-7 days (6.Dickson G. Peck D. Moore S.E. Barton C.H. Walsh F.S. Nature. 1990; 344: 348-351Crossref PubMed Scopus (112) Google Scholar). Using this system, we observed that expression of NCAM precedes the expression of polysialic acid. By transfecting various forms of NCAM, we found that NCAM containing MSD in particular facilitates myoblast fusion and that this activity is diminished by mutation of potential O-glycosylation sites within MSD. On the other hand, abolition of N-linked polysialylation sites in NCAM facilitated myoblast fusion, whereas overexpression of polysialic acid inhibited myoblast fusion. These results indicate that the O-glycans on the MSD of NCAM facilitate myoblast fusion, whereas polysialic acid attenuates this process.EXPERIMENTAL PROCEDURESAntibodies—Anti-mouse NCAM antibody H.28-123 and anti-skeletal myosin heavy chain antibody MY-32 were purchased from Immunotech and Sigma, respectively. Cyan fluorescence protein (CFP) was detected using anti-GFP peptide antibody (Clontech). Human NCAM was stained with anti-CD56 antibody (ERIC-1, Leinco Technologies), which does not react with mouse NCAM. A hybridoma of anti-polysialic acid antibody 5A5 (18.Dodd J. Morton S.B. Karagogeos D. Yamamoto M. Jessell T.M. Neuron. 1988; 1: 105-116Abstract Full Text PDF PubMed Scopus (639) Google Scholar) was purchased from the University of Iowa Hybridoma Bank.Plasmid Construction and Transfection—cDNA encoding human NCAM containing MSD and NCAM without MSD was cloned into pcDNA3.1 as described previously (19.Franceschini I. Angata K. Ong E. Hong A. Doherty P. Fukuda M. Glycobiology. 2001; 11: 231-239Crossref PubMed Scopus (35) Google Scholar). cDNA encoding GPI anchoring domain was excised from human NCAM 120 in pHβAPr-1-neo by KpnI-PvuII digestion and inserted into KpnI-EcoRV sites of pcDNA3.1-NCAM, resulting in pcDNA3.1-NCAM-GPI. The HindIII-EcoRI fragment of NCAM, of which the fourth, fifth, and sixth N-glycosylation site asparagines were mutated to glutamine (15.Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), replaced the corresponding region of pcDNA3.1-NCAM-GPI. By these constructions, we could obtain four isoforms of NCAM (M+N-, M-N-, M+N+, and M-N+). NCAM (M+N+) and NCAM (M-N+) are equivalent to 125- and 120-kDa wild type human NCAM isoforms, respectively.Mutation of the O-glycosylation site threonine to alanine was introduced by PCR using mutated primers: TA1-forward, TCTGCTAGCTCGTCTGCACCTGTTCCATT; TA2-f, TCTGCTAGCTCGTCTGCACCTGTTCCATTGTCTCCACCAGATGCAACT; TA3-f, TCTGCTAGCTCGTCTGCACCTGTTCCATTGTCTCCACCAGATGCAACTTGGCCTCTTCCTGCCCTTGCAGCCACA (NheI site in boldface type; codon for Ala underlined). GPI-reverse, AAGAGTCACCGCAGAGAAAAGC was used as the 3′ primer. The mutated fragment was amplified and introduced into the NheI-KpnI site of pcDNA3-NCAM (M+N-)-GPI.Cloning of cDNAs encoding human ST8Sia II and ST8Sia IV was described previously (11.Nakayama 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, 20.Angata 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). cDNA encoding ST8Sia II and ST8Sia IV fused with CFP was constructed as described (21.Angata K. Yen T.Y. El-Battari A. Macher B.A. Fukuda M. J. Biol. Chem. 2001; 276: 15369-15377Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Briefly, the PstI-AgeI PCR product of ST8Sia IV or ST8Sia II was introduced into the pECFP-N1 vector (Clontech), and then the entire ST8SiaIV-CFP chimera was transferred to pcDNA3. Both immunostaining and immunoblots showed that NCAM was extensively polysialylated by chimeric enzymes, indicating that modification of the C terminus of polysialyltransferases by CFP did not affect their activity as reported for ST8Sia IV-GFP (21.Angata K. Yen T.Y. El-Battari A. Macher B.A. Fukuda M. J. Biol. Chem. 2001; 276: 15369-15377Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).cDNA encoding ST8Sia IV-CFP was introduced to PstI-NotI sites of the pIND vector of the ecdysone-inducible expression system (Invitrogen).Antisense DNA of ST8Sia II or ST8Sia IV was amplified by PCR. cDNAs encoding full-length ST8Sia II or ST8Sia IV were flipped, and KpnI and HindIII sites were attached and then ligated to the N terminus of GFP in pcDNA3.1. Primers used for PCR were as follows: hST8SiaIVa-f, CGGGGTACCAACCAGGACTTTCTCGGGCAC; hST8SiaIVa-r, GTGCCCAAGCTTGTGCATATTGTTTGTTTC; hST8SiaIIa-f, CGGGGTACCGGTCCTGGCGGGCGAACCCACCATG; hST8SiaIIa-r, GTGCCCAAGCTTCTACGTGGCCCCATCGCACT. All cDNA sequences were confirmed by sequencing.Establishment of C2C12 Stable Transfectants—C2C12 cells and rhabdomyosarcoma TE671 and RD cells were obtained from the American Type Culture Collection. C2C12 cells were routinely maintained in DME-high glucose with 20% fetal calf serum. To induce differentiation, the medium was changed to DME-high glucose with 2% horse serum when cells reached confluence, and the medium was changed every day (6.Dickson G. Peck D. Moore S.E. Barton C.H. Walsh F.S. Nature. 1990; 344: 348-351Crossref PubMed Scopus (112) Google Scholar).NCAM or polysialyltransferase plasmids were transfected into C2C12 cells at myoblast stages using LipofectAmine PLUS (Invitrogen) as described previously (20.Angata 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). Stable transfectants of NCAM were selected in hygromycin or G418 (O-glycosylation mutants), and ST8Sia II or ST8Sia IV transfectants were selected by G418. Each stable cell clone apparently exhibited different fusion competency, probably due to difference in the locus of integration of transfected DNA and number of integrated DNA. Also, by immunostaining and immunoblot, we found that expression level of NCAM or polysialic acid varies among clones. We could observe that integrated DNA induced or inhibited fusion depending on transfected cDNAs. To avoid clonal variation, we decided to use a mixture of clones expressing NCAM or polysialic acid.Transfected cells were detached from dishes by cell dissociation buffer (Specialty Media Inc.), and stained with anti-NCAM (ERIC-1) or anti-polysialic acid (5A5) antibody followed by secondary antibody conjugated with FITC. After staining, cells were incubated in DME medium for 20 min at 37 °C to internalize fluorescence-tagged antibodies into cells. The cells were then washed with PBS, digested with trypsin at 37 °C for 8 min, suspended in sorting buffer (5% dialyzed fetal calf serum, 25 mm Hepes, pH 7.4, in PBS), filtered through 40-μm nylon mesh, and sorted by FACStar (Becton Dickinson). This procedure was necessary, since C2C12 cells dissociated without trypsin tend to aggregate. Expression of transfected proteins on sorted cells was confirmed by immunoblot and immunostaining as described previously (22.Yeh J.C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar).ST8SiaIV-CFP/pIND ecdysone-inducible vector and pVgRXR ecdysone receptor were transfected into C2C12 sequentially according to the manufacturer's protocol (Invitrogen). The ST8Sia IV-positive clone was selected by G418 and Zeocin resistance, and cells were stained with anti-CFP antibody before and after induction to confirm expression of ST8Sia IV-CFP. The expression of ST8Sia IV-CFP was induced by addition of the inducer, ponasterone A.Cell Fusion Assay—For each NCAM mutant, three or four independent series of transfection, cell sorting, and cell fusion assays were performed. Cells were plated at a density of 8 × 105 cells on coverslips in each well of 6-well plates, and 40 h after plating, medium was changed from growth (20% fetal calf serum in DME) to differentiation (2% horse serum in DME) medium (6.Dickson G. Peck D. Moore S.E. Barton C.H. Walsh F.S. Nature. 1990; 344: 348-351Crossref PubMed Scopus (112) Google Scholar). After every 24 h, a coverglass was sampled and fixed in 4% paraformaldehyde. From day 0 to 7, a total of eight coverglasses were collected for each cell line. Cells were permeabilized with 0.5% Triton X-100 in PBS for 2 min and blocked with 1% bovine serum albumin in PBS, and the cells were stained with anti-myosin antibody followed by secondary antibody conjugated to fluorescein isothiocyanate or rhodamine, treated with 0.0005% Hoechst 33258 for 5 min. Staining was analyzed using a Nikon ECLIPSE TE300 microscope connected with a SPOT CCD camera or Bio-Rad MRC 1024 confocal laser-scanning microscope. After counting the number of nuclei in four fields for each coverslip at 40 × 10 magnification (∼200 cells in one field), the average ratio (percentage) of nuclei in myosin-positive cells/total nuclei was calculated as a fusion index. The S.D. is shown for a variation among four fields.Immunoblotting—Cells were scraped and lysed in radioimmune precipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) containing protease inhibitor mixture (Sigma) by trituration through 26G⅜-gauge needles. Cell lysates were subjected to sonication in ice-cold water for 15 min and left on ice for another 15 min, and cell debris was removed by brief centrifugation. The protein concentration of cell extracts was determined by a micro-BCA kit (Pierce). Twenty μg of protein was applied to each lane of SDS-PAGE (6% gel) and then blotted onto polyvinylidene difluoride membranes, and conventional immunoblotting was performed.Polysialylated proteins were immunoprecipitated from C2C12 cell lysates or [3H]glucosamine-labeled cell lysates by using 5A5 anti-polysialic acid antibody and goat anti-mouse IgM antibody conjugated to agarose beads (Sigma). The immune complex was washed by radioimmune precipitation assay buffer and PBS and subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting using NCAM antibody (nonradioactive) or fluorography (radioactive).RT-PCR—Following the manufacturer's protocol, 100-200 μg of total RNA was isolated from C2C12 cells using the TRIZOL RNA extraction reagent (Invitrogen). After treatment with DNase I, 20 μg of total RNA was reverse transcribed by SuperScript II (Invitrogen) using an oligo(dT) primer. The cDNA sample was dissolved in 100 μl of water, and 2.5 μl was used for each PCR. Competitive PCR for mouse ST8Sia II and ST8Sia IV was carried out essentially as described with a slight modification (23.Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8: 415-424Crossref PubMed Scopus (122) Google Scholar).Since PCR products for NCAM showed several bands, these bands were separately excised, cloned into TA vector (pGEM-5Zf(+), Promega) and sequenced. Primers used to detect NCAM variants in PCR were as follows: MSD-f, GGTGCAGTTTGATGAGCCAGAGG; MSD-r, GATAGTGTCTGATGGGGGAGCC; GPI/TMD-f, CAGGTAGATATTGTTCCCAGCC; GPI-reverse; TMD-r, AGTCCGTTCTTCCTCCGTTC. PCR using MSD-f and MSD-r yields PCR products from transcripts encoding MSD. PCR using GPI/TMD-f and GPI-reverse yields PCR products for GPI-anchored NCAM, whereas PCR with TMD-f and TMD-r yields products for transmembrane domain-containing NCAM.Oligosaccharide Structure Analysis—C2C12 cells were metabolically labeled with 40 μCi/ml of [3H]glucosamine in 10 ml of glucose-free DME medium for 4 h on day 2, and then 10 ml of DME-high glucose, 2% horse serum was added and incubated for an additional 20 h at 37 °C. Labeled cells were scraped on day 3 and lysed in radioimmune precipitation assay buffer as described above.The same amount of protein derived from control and ST8Sia IV-transfected C2C12 cells was subjected to immunoprecipitation by anti-NCAM antibody (H.28-123). Ten μl out of 300 μl of immunoprecipitate suspension were subjected to SDS-polyacrylamide gel electrophoresis and fluorography, using Amplify (Amersham Biosciences). The rest of the samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After staining polyvinylidene difluoride membrane by Coomassie Brilliant Blue R-250, bands corresponding to each NCAM isoform were excised and subjected to N-glycanase digestion (N-glycosidase F; Calbiochem). Supernatants of the N-glycanase digest were purified and subjected to Mono-Q anion exchange high performance liquid chromatography as described previously (15.Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar).RESULTSNCAM-MSD and Polysialic Acid Are Sequentially Expressed on the Cell Surface of C2C12 Cells—Differentiation of C2C12 cells can be induced by substitution of culture medium containing 20% fetal calf serum with 2% horse serum (day 0, D0). Fig. 1A shows that NCAM and polysialic acid were sequentially expressed on the cell surface after induction of differentiation. Myosin represents a marker of differentiated cells (day 3 and later). Before induction, neither NCAM nor polysialic acid was clearly detected on the cell surface. At the onset of myoblast fusion at around day 1-3, NCAM first appeared on the cell surface, followed by polysialic acid. Fig. 1B shows confocal images at day 5. On mature myotubes, both polysialic acid and NCAM were expressed. Whereas NCAM was expressed mostly at the outer surface of myotubes (see red color), polysialic acid was almost exclusively expressed on the outside surface of myotube bundles (Fig. 1B, green). The latter finding is consistent with previous observation of chick myoblast development that outer surface of myotube bundle is decorated by polysialic acid (24.Fredette B. Rutishauser U. Landmesser L. J. Cell Biol. 1993; 123: 1867-1888Crossref PubMed Scopus (96) Google Scholar). Strong staining of polysialic acid can be occasionally observed on fused cells at day 3, but its function is unknown.Four Isoforms of NCAM Are Expressed in C2C12 Cells—To determine how NCAM expression is regulated during C2C12 cell differentiation, we examined the amount of NCAM and its transcription. First we could observe several isoforms of NCAM protein by immunoblotting (Fig. 2B). The top band corresponds to the TM form of NCAM with MSD (155 kDa), and the middle thick band consists of two bands, the TM form without MSD (140 kDa) and GPI form with MSD (125 kDa). The lower band is the GPI form without MSD. These four isoforms were clearly separated after sialidase digestion (Fig. 2B, second row). The blot of sialidase-treated NCAM shows switching in the synthesis of NCAM from the TM form to the GPI + MSD form as differentiation progresses, consistent with the developmental change of NCAM isoforms in chick embryonic muscle (25.Yoshimi T. Mimura N. Aimoto S. Asano A. Cell Struct. Funct. 1993; 18: 1-11Crossref PubMed Scopus (7) Google Scholar). Lectin blots using PNA, which is specific for O-linked Galβ1-3GalNAc, showed that the GPI + MSD isoform is O-glycosylated during days 2-8 (Fig. 2B). Interestingly, O-glycosylation of TM+MSD and soluble forms of NCAM were also observed. We then examined the transcription of NCAM mRNAs by RT-PCR. RT-PCR analysis confirmed that NCAM was expressed as both GPI-anchoring and transmembrane (TM) isoforms, and that both isoforms contain various forms of MSD insertion (Fig. 2C). At the onset of myoblast fusion at around day 2, MSD began to be transcribed, and the levels of NCAM isoforms with MSD insertion (abcK, abK, and a) gradually increased. To characterize polysialylated NCAM expressed in C2C12 cells, polysialic acid-containing glycoproteins were immunoprecipitated from [3H]glucosamine-labeled C2C12 cells at day 6. The anti-polysialic acid antibody 5A5 immunoprecipitated NCAM and a smaller molecule with relative molecular mass around 40 kDa (Fig. 2D, lane 1). Polysialic acid is attached to N-glycans, since it was released by N-glycanase digestion (Fig. 2D, lane 2). Polysialic acid is mostly attached to the transmembrane form, NCAM-140 (Fig. 2D, lanes 4 and 5). From these analyses, it can be concluded that the GPI+MSD isoform of NCAM is O-glycosylated during the fusion process, whereas polysialylation is restricted to the transmembrane form of NCAM that does not contain MSD.Fig. 2Expression of NCAM muscle-specific domain during in vitro differentiation of C2C12 cells.A, schematic structures of mouse NCAM. MSD is flanked by two fibronectin type III domains (FNIII) and encoded by four exons. Ig, immunoglobulin-like domains. B, total cell lysate was separated by SDS-PAGE before and after sialidase treatment and blotted onto polyvinylidene difluoride membrane. The blot was reacted with anti-NCAM antibody. O-glycans of immunoprecipitated NCAM were visualized using PNA. NCAM containing TM, MSD, GPI-anchored NCAM, and soluble NCAM (sol) are shown. C, RT-PCR of MSD and GPI-anchored and TM-containing NCAM during differentiation of C2C12 cells. abcK, abK, acK, a, and no correspond to those in A, D, lanes 1-3, C2C12 cells were metabolically labeled with [3H]glucosamine at days 5-6 and immunoprecipitated with anti-polysialic acid antibody. Aliquots were treated without (lane 1) or with N-glycanase (lane 2) or with endoneuroaminidase-N (lane 3). Fluorography of [3H]-glucosamine-labeled proteins after separation on SDS-polyacrylamide gels is shown. Lanes 4 and 5, total proteins of C2C12 cells immunoprecipitated with anti-polysialic antibody (lane 4) or total cell lysate (lane 5) were separated on SDS-PAGE, blotted, and reacted with anti-NCAM antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Abolition of MSD and Polysialic Acid on NCAM Differentially Alters Myoblast Fusion Rate—To examine the effect of NCAM glycosylation on myoblast fusion, we constructed NCAM mutants lacking N-glycosylation sites that are preferentially polysialylated, or O-glycosylation sites in the MSD (Fig. 3A). In our previous study, we found that the fourth, fifth and sixth N-glycosylation sites of NCAM are preferentially polysialylated (15.Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). On the other hand, from the results shown above (Fig. 2B) and from previous reports (4.Walsh F.S. Parekh R.B. Moore S.E. Dickson G. Barton C.H. Gower H.J. Dwek R.A. Rademacher T.W. Development. 1989; 105: 803-811PubMed Google Scholar, 26.Ong E. Suzuki M. Belot F. Yeh J.C. Franceschini I. Angata K. Hindsgaul O. Fukuda M. J. Biol. Chem. 2002; 277: 18182-18190Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), it can be concluded that O-glycans are attached to the MSD. Therefore, based on the structure of GPI anchoring isoforms present in nature, we constructed cDNAs encoding chimeric proteins of NCAM with and without N-glycosylation sites for polysialylation and with and without the MSD (Fig. 3A). Stable cell lines expressing each NCAM isoform were established, and the expression of transfected human NCAM was confirmed by immunoblot (Fig. 3B) and fluorescence-activated cell sorting analysis. The results shown in Fig. 3C indicate that C2C12 cells expressing NCAM+MSD (M-) tend to fuse faster, forming elongated mature myotubes at day 5, than cells expressing NCAM without MSD (M-) when they do not contain polysialylated N-glycans (N-). By contrast, C2C12 cells expressing polysialylated NCAM (N+) tend to fuse more slowly, and fused cells form round and short myotubes. Graphical representation of these results (Fig. 3D) indicates that C2C12 cells expressing NCAM containing the MSD (M+) showed enhanced fusion, whereas this MSD activity is not prominent when N-glycan polysialylation sites are intact (N+). These results clearly demonstrate that the MSD on NCAM has a positive effect, whereas have N-glycans containing polysialic acid on NCAM have a negative effect on myoblast fusion.Fig. 3Effect of MSD and N-glycan polysialylation on C2C12 cell differentiation.A, schematic structure of NCAM with MSD (M+) or without MSD (M-) or w
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