CELL STRUCTURE AND FUNCTION 19: 241-252 (1994)

© 1994 by Japan Society for Cell Biology

Selective Inhibition of a Step of Myotube Formation with Wheat Germ Agglutininin a Murine Myoblast Cell Line, C2C12

Siisiimu Muroya, Hidekazu Takagi, Shoji Tajima*, and Akira AsanoInstitute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565, Japan

Key words: myotube/myogenesis/wheat germ agglutinin/cell fusion

ABSTRACT.Myoblast cells, C2C12, which is an established cell line from satellite cells of skeletal muscle ofC3Hmouse, start to fuse and form multinucleated cells (myotubes) and begin to express creatine phosphokinaseand myosin, whenculture mediumis changed from the growth mediumto the differentiation medium.Amongthe 12 lectins that we tested, wheat germ agglutinin apparently suppressed the myotube development judged byphase-contrast microscopy, but did not affect the induction of creatine phosphokinase activity. The addition ofN-acetylglucosamine or N,N',N"-triacetylchitotriose, which is a specific ligand for wheat germ agglutinin, to thedifferentiation medium,recovered this apparently suppressive effect of wheat germ agglutinin on the myotubedevelopment. Tachypleus tridentatus (Japanese horseshoe crab) lectin that specifically recognizes N-acetylneu-raminic acid, one of the sialic acids, showedno effect on the myotubedevelopment. It was suggested that wheatgerm agglutinin suppressed the process through recognizing N-acetylglucosamine containing sugar. Surprising-ly, even in the presence of wheat germ agglutinin, the ratio of mononucleated cell numbers to the genomic DNAcontent, which represents the fusion level, decreased after incubation in the differentiation medium, indicatingthat even when wheat germ agglutinin was present in the medium, cell fusion, which is the initial step of the myo-tube formation, occurred. Immunostaining with anti-skeletal muscle myosin antiserum confirmed that the myo-sin expressing cells actually fused and formed multinucleated cells. Their shape, however, was thin compared tothat in the absence of wheat germ agglutinin. Wepropose that the membranefusion step to form myotubes iscomposed of two distinct steps in C2C12; one fusion step is to form long and thin myotubes from mononucle-ated cells and the other one is to develop fat myotubes. Wheat germ agglutinin specifically inhibits the latter fu-sion step.

During the terminal differentiation of myogenesis,myoblasts fuse to form multinucleated myotubes. Todate, several proteins that are involved in the process ofmyoblast fusion have been reported. Neural cell adhe-sion molecule (N-CAM)and N-cadherin are expected tofunction in the initial recognition step for the fusion ofembryonic chicken myoblasts (1-4). However, the spe-cific antibodies against the proteins do not inhibit themyoblast fusion efficiently (1, 3-5). Both VLA-4 (in-tegrin a4/31) and vascular cell adhesion molecule 1

(VCAM-1)play important roles in the cell to cell recog-nition at the stage of secondary myoblast fusion toform mature myotubes in vivo (5). The monoclonal anti-body raised against either VLA-4 or VCAM-1effective-ly inhibits the myotube formation of C2C12 cells (5). Be-sides these four proteins, many other proteins are re-ported to be involved in the fusion of myoblasts (6-1 1).Myoblast fusion is expected to be controlled by pro-teins localized on the cell surface. Manyof them are gly-coproteins. Tunicamycin, an inhibitor for protein glyco-sylation, inhibits myotube formation (12, 13). Actually,almost all the proteins that are expected to play roles inthe myoblast fusion described above are glycoproteins.Therefore, the lectins that recognize specific structureof the sugar chain are useful tools to examine the myo-blast fusion. At present, concanavalin A (ConA) andwheat germ agglutinin (WGA) are known to inhibit thefusion of primary culture of chicken myoblasts (14).The inhibitory effect of ConAwas not specific on themyoblast fusion but on the entire myogenicprocess ofthe cells (15, 16).

We tested the effect of 12 lectins on the myotube for-

* Towhomcorrespondenceshouldbe addressed.Abbreviations: N-CAM,neural cell adhesion molecule; VCAM-

1, vascular cell adhesion molecule 1; ConA, concanavalin A; WGA,wheat germ agglutinin; DBA, Dolichos biflorus lectin; LCA,, Lensculinaris agglutinin; MAM,Maackia amurensis lectin; PNA, peanutlectin; RCA-120, Ricinus communis lectin 120 (RCA-120); SBA, soy-bean lectin; SSA, Sambucus sieboldiana lectin; UEA-I, Ulex europae-us lectin-I; AAL, Aleuria aurantia lectin; TTA, Japanese horseshoecrab lectin; DMEM,Dulbecco's modified Eagle's medium; FBS, fetalbovine serum; TAC, N,N',N"-triacetylchitotriose; MCK,muscle crea-tine phosphokinase; PBS (-), calcium-magnesium free Dulbecco'sphosphate buffered saline.

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mation in C2C12cells, and found that one of the testedlectins, WGA,specifically suppressed the developmentof the myotubes without affecting the initial myoblastfusion.

MATERIALS AND METHODS

Materials.Dulbecco's modified Eagle's medium (DMEM)was pur-chased from Nissui Seiyaku (Tokyo, Japan), and Cosmediumwas from CosmoBio (Tokyo, Japan). Fetal bovine serum(FBS) and G418 were obtained from Gibco BRL Japan(Tokyo). Trypsin was from DIFCO (Detroit, Michigan), andgelatin was from Iwaki Glass (Tokyo, Japan). ConA, Doli-chos biflorus lectin (DBA), Lens culinaris agglutinin (LCA),Lotus tetragonolobus lectin (lotus lectin), Macckia amurensislectin (MAM), peanut lectin (PNA), Ricinus communis lectin120 (RCA-120), soybean lectin (SBA), Sambucus sieboldianalectin (SSA), Ulex europaeus lectin-I (UEA-I), and WGAwere purchased from Honen Corp. (Tokyo, Japan). Aleuriaaurantia lectin (AAL) and Japanese horseshoe crab lectin

(TTA) were from Nichirei (Tokyo, Japan) and Seikagakuko-gyo Corp. (Tokyo, Japan), respectively. N,N',N"-triacetylchi-totriose (TAC), penicillin, and streptomycin were obtainedfrom Sigma (St. Louis, Missouri). Hoechst 33258 and goat an-ti-rabbit antibody-peroxidase were obtained from WakoPureChemicals (Osaka, Japan). The other chemicals used were of

the highest quality available.

Methods.

Cells and Cell Culture. Mouse skeletal muscle myoblastcell line, C2C12, and its transformant, MTR4C (17), weremaintained as monolayers in a humidified incubator (5%

CO2) at 37°C in plastic dishes that were coated with 1% gela-tin, containing DMEMsupplemented with \0% FBS, 100 u/ml of penicillin, and 100 /ig/ml of streptomycin. As for main-taining MTR4C, 4-5 mg/ml of G418 in 0.1 M Hepes-NaOH,pH 7.4, was added at 0.2 mg/ml to the culture medium. Twodays prior to the experiment, samples of 1x105 cells ofC2C12 or 2 x 105 cells ofMTR4Cwere seeded into 60-mm plas-tic dishes containing 3 ml of growth medium. On day 2 of cellgrowth, the growth mediumwas replaced by 3 ml of Cosmedi-um, the differentiation inducing medium (18). Every 24 h, thedifferentiation mediumwith or without appropriate concentra-tion of lectin was changed for the fresh one. Each lectin wasadded to the differentiation mediumat various concentrations(0.2-10 /ig/ml) after 12 or 6 h incubation, and the incubationwas continued for 72 or 48 h for C2C12 or MTR4C, respective-ly (Fig. 1). The conditions were used for all the studies de-scribed hereafter unless otherwise stated.Measurement of muscle creatine phosphokinase (MCK)activity. The cells cultured in 60-mmplastic dishes werewashed twice with calcium-magnesium free Dulbecco's phos-phate buffered saline (PBS (-)) and lysed with 1 ml of ice

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Fig. 1. Schematic illustration of time table of cell culture. The culture medium for C2C12 or MTR4Ccells was switched to the differentiationmedium (Cosmedium) at time 0. Cosmedium was changed with fresh one with or without lectin every 24 h. The closed arrows indicate the timepoints at which lectins were added.

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Inhibition of Myogenesis by Wheat Germ Agglutinin

cold cell lysis buffer (1 M NaCl, 1 mMEGTA, 1% Triton-X100, 10 mMTris-HCl, pH 7.2) (19). The lysate, after freez-ing and thawing, was briefly (about 5 seconds) sonicated at 50Win a Branson Sonifier equipped with a micro tip, and thencentrifuged at 20,000 x g at 4°C for 15 min. The supernatant

fraction served for the measurement of MCKactivity (20, 21).The protein concentration of the cell extract was determinedby BCAprotein assay kit (Pierce) using bovine serum albuminas a standard.Determination of the mononucleated cell numbers and the

Fig. 2. Effects ofWGAon the myotube development. C2C12 cells (A) and MTR4C(B) were cultured in the growth medium. The medium wasthen changed to the differentiation medium and the culture was continued for 72 h for C2C12 (C, E), and 48 h for MTR4C(D, F), with (E, F) orwithout (C, D) WGA(2 /*g/ml). The bar indicates 100 /mi.

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total DNAcontent. The C2C12 cells in 60-mm dish werewashed with PBS (-), and dissociated with 1 ml of 0.25%trypsin and 0.02% EDTAat 37°C for 15 min. The cells weresuspended and the cell numbers were counted in a hemocytom-eter. A small portion of the cell suspension was fixed with3.7% formaldehyde on coverglass coated with poly-L-lysine(Sigma) for 10 min. The fixed cells were stained with Mayer-haematoxylin (MERCK),and the numbers of nuclei per cellwere counted. More than 99% of the counted cells containedone nucleus. From the rest of cell suspension, the genomicDNAwas isolated as described previously (22). The genomicDNAthus prepared was dissolved in 0.5 ml of 10mMTris-HC1, 1 mMEDTA(pH 8.0) and was briefly sonicated. An ali-quot of this fraction was added to 200 /ig/ml of Hoechst33258 to a final concentration of 1 ^g/ml (23). The reactionmixture was excited at 356 nmand an emission at 456 mmwasmeasured. Salmonsperm DNAwas used as a standard.Immunostaining of Myotube Using Anti-myosin Antise-rum. The cells induced to differentiate were washed with icecold PBS (-) and fixed with methanol at -20°C for 5 min.The cells were then washed with PBS(-), and soaked with3% H2O2 for 5 min to inactivate endogenous peroxidase. Thecells were washed again with PBS (-) and incubated with rab-bit anti-myosin antiserum in the antibody buffer (1% bovineserum albumin and 0.02% sodium azide in PBS (-)) at roomtemperature for 90min. The antiserum used was raised

against mouse skeletal muscle myosin heavy chain. After the

Table I. Effects of lectins on the myotube formationAND THE MCK ACTIVITY.

Jgt?l) -**ub, MCgVky

WGA (2 /ig/ml) - 67I AAL (10 /ig/ml) - 86

LCA (10 /ig/ml) - 150

ConA (5 /ig/ml) + 122DBA (10 /ig/ml) + 84

SBA (10 /ig/ml) + 88PNA (10 /ig/ml) + 98

Lotus (10/ig/ml) + 105UEA-I (10 /ig/ml) + 85

RCA120 (0.2 /ig/ml) - 0III SSA (2 /ig/ml) - 24

MAM (10/ig/ml) - 0

After 48 h incubation in the differentiation medium, the myotubeformation was judged and the MCKactivity was determined as de-scribed in the "MATERIALSANDMETHODS".When no "typical"myotube was observed, the myotube formation was expressed as (-).Whenmorethan one myotubewas found in a dish, the myotubefor-mation was expressed as (+). The MCKactivity was expressed as per-cent of the specific activity of the C2C12cells cultured in the absenceof lectin. The lectins were classified into three groups: I, myotube (-)and MCKnormal; II, myotube (+) and MCKnormal; III, myotube(-) and MCK induction inhibited.

incubation, the cells were washed with PBS (-). Next, thecells were incubated with anti-rabbit antibodies conjugated toperoxidase in PBS (-) at room temperature for 30 min, andthen washed with PBS (-). The bound second antibodieswere visualized using a color reaction buffer containing 0.4mg/ml 3-amino-9-ethylcarbazole, 0.03% H2O2 in 50 mMsodi-um acetate, pH 5.3. Nuclei DNAwas stained with Mayer-hae-matoxylin according to the manufacturer's protocol (DaiichiPure Chemicals; Tokyo, Japan).

RESULTS

WGAapparently suppressed the myotube develop-ment. Whencultured myoblasts are induced to differ-entiate, the cells start to fuse and form multinucleatedcells. Wetested the effect of 12 lectins on the myotubeformation of C2C12 and MTR4Caccording to the timetable shown in Fig. 1. MTR4Cis established by trans-fecting the mouse DNAmethyltransferase CDNAtoC2C12 (17), of which myotube formation including theinduction of MCKand myosin is about 24 h earlier thanthat of the parent C2C12. After 72 or 48 h culture in

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Fig. 3. The induction ofMCKactivities in C2C12 cells after the me-dium was changed to the differentiation medium, in the presence orabsence of WGA(2 ^g/ml) and/or GlcNAc (20 mM). The closed ar-row indicates the time when WGAand/or GlcNAc were added. Thecells cultured in Cosmediumwere harvested at the indicated time peri-ods, and the MCKactivities and the amount of proteins were deter-mined as described in the "MATERIALSANDMETHODS".The ac-tivities were demonstrated as specific activities (units//zg protein). Theopen arrow indicates the time point at which photographs shown inFig. 4A-D were taken.

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Inhibition of Myogenesis by Wheat Germ Agglutinin

Fig. 4.

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Fig. 5. Effect of TTA on the myotube development of MTR4Ccells. The cells were cultured without lectin (A), with 2 /ig/ml of WGA(B), orwith 10 ^g/ml of TTA (C). The micrographs were taken after 48 h incubation in the differentiation medium. The bar indicates 100 fim.

Cosmedium, C2C12 or MTR4Ccells clearly formedmultinucleated myotubes (compare Fig. 2 panels A andB with C and D). In contrast, apparently, no typical my-otube was observed when WGA(2 /ig/ml), AAL (10 [ig/ml), LCA (10 /ig/ml), RCA-120 (0.2 /ig/ml), SSA (2/ig/ml), or MAM(10 /ig/ml) was added to Cosmedium.ConA (5 jug/ml), 10 /ig/ml each of DBA, SBA, PNA,lotus lectin, or UEA-I showed no effect on the myotubeformation (summarized in Table I). When 10 ^g/ml ofConA was added to the medium,the cells were apt todissociate from the dish. Typical shapes of the cells inthe presence of WGA(2 fig/ml) are shown in Fig. 2Eand F. At the concentration of 10 or 0.4 ^g/ml, WGAsuppressed the cell proliferation or showed no effect, re-spectively (data not shown).MCK,a skeletal muscle specific enzyme, is induced toexpress by changing the medium to the differentiation

medium. All of the lectins, ConA, DBA, SBA, PNA, lo-tus lectin, and UEA-I, that did not inhibit the myotubeformation, also had no effect on the induction of MCKactivity (Table I, group II). The three lectins, RCA-120,SSA, and MAMapparently inhibited both the myotubeformation and the MCKactivity (Table I, group III).RCA-120 and MAMsuppressed the cell growth. SSA in-hibited the MCKactivity, but had no effect on the cellgrowth. The effect of the lectins on the myotube forma-tion in the group III may be complicated or indirect. In-terestingly, WGA,AAL, and LCA, which apparentlysuppressed the myotube development, showed littleeffect on the MCKactivity (Table I, group I). The lec-tins in group I seemed to inhibit specifically the multinu-cleation step of the myotubeformation without affect-ing the biochemical differentiation. However, AALandLCAmade the cells aggregate. Therefore, we next exam-

Fig. 4. The ligands compete for the suppressive effect of WGAon the myotube development in C2C12. GlcNAc (20 mM)(C and D) or TAC(200 fiM) (G and H) was added to the culture medium. WGA(2 /ig/ml) was added (B, D, F, and H) but not (A, C, E, and G) to the medium. Themicrographs were taken at 60 h (A-D) or 72 h (E-H) after the medium was switched to Cosmedium. The bar indicates 100 ptva.

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Inhibition of Myogenesis by Wheat Germ Agglutinin

ined the action of WGA.The MCKactivity started to increase after 24 h in-cubation in the differentiation medium and rapidly in-creased thereafter (Fig. 3). The increasing profile of theMCKactivity in the presence of WGAwas almost identi-cal to that in the absence of WGA.It was confirmedthat WGA,which apparently suppressed the myotubedevelopment, showed little effect on the MCKactivity.WGAspecifically recognizes and binds to N-acetyl-glucosamine (GlcNAc), oligo-saccharides containingGlcNAc (24, 25), and sialic acids (26). As shown in Fig.3, GlcNAc did not affect the MCKinduction. If the in-hibitory effect of WGAwas due to the specific bindingto the cell surface, GlcNAc and its trisaccharide, TAC,which are the specific ligands for WGAwith dissocia-tion constants of 0.76 mMand 12 fiM, respectively (24),should restore the suppression of myotube develop-ment. In fact, three days after the mediumwas changedto Cosmedium, C2C12 cells showed normal myotubeswhen WGAwas added with 20 mMGlcNAc or 200 fiM

TAC (Fig. 4). WGA is also known to bind to sialicacids. Wetested whether or not Japanese horseshoecrab lectin (TTA), which is known to recognize N-ace-tylneuraminic acid (one of the sialic acids), also sup-pressed the myotube development. WGA(2 fig/ml)clearly inhibited the myotube development of MTR4C,while TTA (10 /ig/ml) had no effect on that step (Fig.5). Therefore, it was strongly suggested that WGAsuppressed the myotube development by recognizingGlcNAc containing sugar.WGAdid not actually inhibit the myoblast fusion.Membrane fusion is the initial step for the myotube for-mation. However, it was difficult to accurately quantifythe step. To evaluate the extent of the fusion of C2C12cells, the ratio of the numbers of mononucleated cells tothe total DNAcontent (=amount of nuclei) was meas-ured. Whenthe cells start to fuse, the parameter is ex-pected to drop. The mononucleated cells were dissoci-ated with trypsin and EDTAand counted in a hemocy-tometer. After the mediumwas changed to the differen-

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Fig. 6. Quantification of cell fusion in the process of myotube formation. The changes in the numbers of the mononucleated cells (A), theamount of DNA(B), and the ratio of the numbers of mononucleated cells to the amount of the DNA(C), during the cell differentiation in the pre-sence ( å¡ ) or absence (O) ofWGA(2 /ig/ml) were demonstrated. C2C12 cells were trypsinized at the indicated time periods, the numbers of mon-onucleated cells counted, and the content of DNAdetermined as described in the "MATERIALSANDMETHODS".

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S. Muroya et al.

tiation medium, in the presence or absence of WGA,the numbers of the mononucleated cells and the

amount of DNAper dish were measured. After 36 h in-cubation, the cell numbers reached twice that of time 0and decreased thereafter (Fig. 6A). The DNAcontent in-creased as well, but did not drop after 36 h incubation(Fig. 6B). As a result, the ratio of cell numbers to DNAcontent dropped after 36 h incubation (Fig. 6C). Multi-nucleation (=cell fusion) occurred actively at this peri-od. This was supported by morphological observations.This, surprisingly, was also observed for the cells added

to which WGAwas added (Fig. 6). The result clearly in-dicates that the addition of WGAto the differentiationmediumdid not inhibit the cell fusion, the initial step ofthe myotube formation.Wenext examined the expression of skeletal musclemyosin in the cells cultured in the presence of WGA.Ifthe myoblasts in the presence of WGAwere really fusedand formed multinucleated cells, visualization of theskeletal muscle myosin by immunostaining would makeit celar. Immunostained C2C12 and MTR4Ccells withanti-myosin antiserum are shown in Fig. 7. Both C2C12

Fig. 7. Effect of WGAon the myotube development and the myosin expression. The cell samples were incubated in the presence (C, D, F) orabsence (A, B, E) of 2 ,ug/ml of WGAin Cosmedium. C2C12 (A, C) and MTR4C(B, D-F) cells were stained with anti-myosin antiserum raisedagainst mouse skeletal muscle myosin heavy chain, after 72 h (A, C) and 48 h (B, D-F) incubations, respectively. MTR4C(E, F) cells were alsostained with Mayer-haematoxylin after 48 h incubation. The bar indicates 100 /urn.

248

Inhibition of Myogenesis by Wheat Germ Agglutinin

00 \( W-iI

\JTV à" WGA /

Con fluentFig. 8. Schematic illustrations of the process of the myotube development in C2C12 cells. After the induction of differentiation, initially, myo-blasts fuse each other to form long and thin myotubes. The thin myotubes then fuse to each other or with myoblasts to form flat and extendedshape. WGAinhibits the second step of the myotube formation.

and MTR4Ccells expressed myosin in the presence of 2fjtg/ml of WGA(Fig. 7C and D). The expressed skeletalmuscle myosin was found in the myotubes that werevery thin but their length was as long as those formed inthe absence of WGA.Thus, it was confirmed that WGAdid not inhibit the myoblast fusion. The cells stainedwith anti-myosin antibody and haematoxylin showedthat the very thin myotubes found in the presence ofWGAactually contained multiple nuclei (Fig. 7F). Inter-estingly, the numbers of non-fused cells that did not ex-press myosinin the presence or absence of WGAweresimilar (Fig. 7E and F, and refer to Fig. 6A). The

shapes of the myotubes in the presence or absence ofWGAwere similar to each other until 21 h incubationin Cosmedium (data not shown), and the inhibitory

effect of WGAbecame clear only after the 48 h incuba-tion (Fig. 7E and F).

DISCUSSION

In the present study, we tested the effect of 12 lectinson the myotube formation of C2C12cells. Amongthelectins tested, WGAsuppressed the myotube develop-ment, but apparently had no effect on the MCKindue-

tion. Although WGAseemed to suppress the myotubedevelopment, the index for the fusion (Fig. 6C) and theimmunostaining of the cells with anti-skeletal musclemyosin antiserum (Fig. 7) revealed that WGAdid not in-hibit the initial step of the process. The inhibitory ac-tion of WGAwas on the development of the fused myo-tubes into large and extended ones. The myotubesformed in the presence of WGAcontained less nucleiper length than those in the absence of WGA(Fig. 7E

and F). Wepropose that the cell fusion process to formmyotubes is composed of two distinct steps; one is to

form long and thin myotubes and the other is to formfat and extended myotubes, which is schematically illus-trated in Fig. 8. At present, we do not have direct evi-

dence that the cell fusion along the long axis to form thethin myotube precedes that of the fusion along theshort axis, as shown in Fig. 8. However, significant

amounts of the long and thin myotubes were found inthe immunostained MTR4Ccells after 48 h incubationeven in the absence of WGA(Fig. 7B). It seems likelythat the myoblasts that are in contact with the end ofthe long axis fuse first to form long and thin myotubes,

and that the myoblasts or the other thin myotubes thenfuse to the primary myotubes to form the fat and ex-

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Fig. 9. Effect of anti-VLA4 antibody on the myotube development in MTR4C.The cells were incubated without (A), or with 2 //g/ml of WGA(B), 25 /ig/ml (C), or 50 /ig/ml (D) of PS/2 in Cosmedium. The antibody was added after 6 h incubation in Cosmedium. The micrographs weretaken after 48 h incubation. The bar indicates 100 ^m.

tended shape.As to be molecule targeted by WGAto inhibit thesecondary fusion of the myoblasts, GlcNAc (24) andsialic acids (26) are reported to be the specific ligandsfor WGA.Actually, GlcNAcand its trisaccharide sup-pressed the inhibition of WGA(Fig. 4), and Japanesehorseshoe crab lectin that specifically binds to N-acetyl-neuraminic acid (27), one of the sialic acids, showed noeffect on the myotube formation. Therefore, it was sug-gested that WGAinhibited the myotube developmentthrough recognizing GlcNAc containing sugar.At present, four membrane glycoproteins, N-CAM(1, 2, 4), N-cadherin (3), VLA-4 (integrin a4/31), and

VCAM-1(5), are reported to be involved in the myogen-esis. N-CAMutilizes muscle specific exons when thecells differentiate into myotubes (28-30), and the intro-duction of the CDNAcontaining this muscle specific do-main into the myoblasts promotes the myotube forma-tion (3, 4). The specific antibody against the moleculeproduced from the introduced CDNApartially sup-

presses the myogenesis (3, 4). However, peanut lectin(PNA), which specifically binds to the 0-linked glycosyl-ation site of the muscle specific domain (31), did not in-hibit the myotube formation of C2C12 (Table I). N-CAMmight not be the target of WGA.On the otherhand, 100 //g/ml of anti-VLA-4 (integrin a4/31) mono-clonal antibody, PS/2, or anti-VCAM-1 monoclonal an-tibody, MK/1, inhibits the myoblast fusion of C2C12very efficiently (5). In another study 100 fig/ml of themonoclonal antibody PS/2 (generously provided byDr. Miyake at Saga Medical School) (32) inhibited notonly the myoblast fusion, but also the expression ofmyosin (data not shown). Less than 10 fig/ml of PS/2showed no effect on the myotube formation, but 25 or50 fig/ml of PS/2 partially inhibited the developmentof the myotube formation, similar to that of 2 fig/mlof WGA(Fig. 9). The myosin expression was inducedin the presence of 25 or 50ptg/ml of PS/2 (data notshown). In addition, the receptor for fibronectin,knownas a memberof integrin family, is reported to

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Inhibition of Myogenesis by Wheat Germ Agglutinin

bind to WGA(33, 34). It is reasonable to speculate thatVLA-4is the best candidate of the target of WGA.Adiscrepancy exists however, since in C2C12, aA subunitof VLA-4is not expressed in myoblasts but in the cellsthat are induced to differentiate (5), while, in the pre-sent study, WGAhad to be present at very beginning ofthe induction of the differentiation for the inhibition.WhenWGAwas added to C2C12 cells after 24h in-cubation in Cosmedium, the added WGAcould notsuppress the myotube development anymore (data notshown). Further experiments are needed to determinewhich molecule is the target of WGA.

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184.Dickson, G., Peck, D., Moore, S.E., Barton, C.H., and

Walsh, F.S. 1990. Enhanced myogenesis in NCAM-trans-fected mouse myoblasts. Nature, 344: 348-351.Peck, D. and Walsh, F.S. 1993. Differential effects of over-ex-pressed neural cell adhesion molecule isoforms on myoblast fu-

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{Received for publication, March 29, 1994and in revised form, May 23, 1994)

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