Purified lectin from skeletal muscle inhibits myotube formation in vitro.

A lactose-extractable lectin obtained from 14--16-d embryonic chick pectoral muscle and myotube muscle cultures by affinity chromatography inhibited myotube formation in culture. When applied to muscle cultures at 0.09 micrograms/ml, the purified lectin produced variable effects on the inhibition of myotube formation related to the time and length of application, suggesting that components of the culture medium and/or temperature produced inactivation. Hemagglutination assays showed that the lectin was inactivated by horse serum and by chick embryo extract but not by L-15 salt solution at 4 degrees C. Incubation in L-15 solution at 37 degrees C with or without 2 mM dithiothreitol resulted in inactivation in 2--3 h. To maximize the effect of the lectin on the inhibition of myotube formation, primary muscle cultures were grown in low [Ca+2] medium to inhibit fusion, and then [Ca+2] was increased to elicit fusion in the absence and presence of lectin with solution renewal every 2 h. Without lectin, myotube formation was normal, whereas, with lectin, it was inhibited by 93%. Continued incubation at 37 degrees C. without renewal of lectin resulted in myotube formation, suggesting reversibility by lectin inactivation.

It has been postulated that embryonic cells employ the appearance and/or disappearance of surface constituents during their development for specific tissue recognition (17,24,29) . Evidence for the appearance of such factors has been obtained for retinal cells and, more recently, for retinal cell membranes that show specificity for type, developmental stage, and absence of recognition factors in older embryos (8,9) . Multivalent glycoproteins that promote species-specific sponge cell aggregation (10) and molecules that appear to be the receptors for these ligands on the cell surfaces (28) have been isolated in pure form . Simpson et al . (25) have characterized and purified a galactosebinding protein, discoidin, from the slime mold Dictyostelium discoideum in which the biosynthesis closely parallels the development of cell cohesiveness . Species-specificity is suggested because an-J . CELL BIOLOGY © The Rockefeller University Press -0021-9525/80/06/0617/09 $1 .00 Volume 85 June 1980 617-625 other slime mold, Polysphondylium pallidum, also contains a developmentally regulated cell surface lectin that reacts with species-specific cell surface oligosaccharides during cellular cohesion (26) .
A /3-D-galactoside-specific lectin occurs in embryonic chick pectoral muscle and skeletal muscle cultures (4,7,19,27). The highest concentration appears between 8 and 16 d of development (19), which is also the period of highest myoblast fusion, suggesting a possible correlation between the lectin and the recognition-fusion process. The hemagglutinating activity of this lectin is inhibited by lactose and thiodigalactoside . Primary cultures of chicken (6) and rat skeletal (4) muscle are unaffected by thiodigalactoside ; however, the rat muscle cell lines L 6 (4, 7) and L 8 (4) are fusion inhibited .
The objective of this study was to determine the effect of direct application of the lectin to skeletal muscle cultures on cell recognition-myoblast fusion. Application of crude extracts to monolayer primary cultures of chicken skeletal muscle showed a partial inhibition of myotube formation . Therefore, we purified the lectin by affinity chromatography, determined its unit molecular weight, and optimized the conditions for employing the lectin in culture to better test the effect on myotube formation. Our studies show that under conditions which minimized inactivation of the lectin, application in picomole concentrations inhibits myotube formation by >90% . While this study was in progress, Den and Malinzak (4) and Nowak et al. (20) reported the isolation of a similar lectin from chicken embryonic muscle. However, its effects on myotube formation have not been reported .

Preparation of Purified Lectin
Extracts of chicken embryonic skeletal muscle and muscle cultures were prepared according to Nowak et al . (19). Pectoral muscles from 14-16-d embryos (20 g wet wt) were homogenized in an Omnimixer (DuPont Instruments-Sorvall, DuPont Co., Newtown, Conn .) in 0.15 M NaCl, 2 mM EDTA, and 2 mM dithiothreitol (DES)' with 0.3 M lactose, pH 8.0, at top speed for I min at 0°C andcentrifuged for I h at 100,000 g. The supernate was dialyzed two times against I liter of DES without lactose for 12 h at 4°C, then two times against phosphate-buffered saline (0 .15 M NaCl and 5 mM NaP0 4, pH 8.0 [PBS]) plus 2 mM dithiothreitol (DTT). Samples were frozen at -20°C until used . Primary myotube cultures (5-d) were rinsed three times with 4°C PBS (7 ml), scraped from the plates with a rubber policeman in DES plus 0.3 M lactose, extracted overnight at 4°C, and centrifuged at 100,000 g. The supernate was dialyzed as described for muscle extracts against DES and PBS plus DTT.
Affinity chromatography was performed as described by Baues and Gray (1) using aminoethyl Bio-Gel P-150 (Bio-Rad Laboratories, Richmond, Calif) coupled with lactose. Coupling was accomplished by a direct covalent attachment of reducing carbohydrates to an aminoethyl polyacrylamide gel via a reductive amination with cyanoborchydride at pH 9 proximately I mg of lectin was obtained per 20 g wet wt of dissected muscle. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the lectin's purity by the method of Kedinger et al . (14) using denaturing (1 .25% SDS) and reducing (1 .25% Q-mercaptoethanol) conditions with a polyacrylamide gradient of 5-17 .5%. Electrophoresis was run for 8 h at 100 V using 3 hg of purified lectin . Molecular weight was determined by passing lectin over a Sephadex G-75 superfine chromatography column usingcytochrome c (12,400 daltons), ovalbumin (45,000 daltons), and bovine serum albumin (BSA) (67,000 daltons) as standards.

Assayfor Lectin Activity
The hemagglutination assay was modified from that of Lis and Sharon (16) . Human blood (type B positive) erythrocytes were prepared fresh for each assay by washing three times with cold PBS, trypsinizing for 1 h at 37°C with a 0.05% trypsin solution containing Eagle's minimal essential medium (MEM) with Spinner's salts, washing three more times with cold PBS, and making a stock 1% (vol/vol) suspension in PBS. The assay was performed by serial twofold dilutions of the lectin with PBS. The dilutions were mixed with an equal volume of the erythrocyte suspension in a microtiter plate with V-shaped wells (Falcon Labware, Div. of Becton, Dickinson & Co ., Oxnard, Calif.) . The cells were allowed to settle for l-2 h, and the titer was determined from the highest dilution showing aggregation, Specific activity wasexpressed as the titer`/mg of protein. Protein determinations were done by the method of Bradford (2) using graded concentrations of BSA as standards.

Skeletal Muscle Cultures
Primary breast muscle cultures from 12-d White Leghorn chicken embryos obtained from the Case Western Reserve University farm were prepared and grown as described by Sandra et al. (22) in 2.5 ml of L-15 medium supplemented with 15% horse serum (HS), 5% chick embryo extract (CEE), and 1% antibioticantimycotic (Grand Island Biological Co ., Grand Island, N. Y.) . To obtain cultures in which myoblast fusion was suppressed, [Ca"] was decreased by dialyzing L-15 medium and HS against Eagle's MEM with Spinner's salts (23) (CEE was not dialyzed to reduce [Ca"]) . Cultures were plated for the first 24 h in fully supplemented medium, and then themedium was changed to the low [Ca"] medium . At the time of testing, lectin was added with supplemented medium to promote myoblast fusion by the increased [Ca"]. Identical cultures with no lectin were maintained, and the medium from these cultures was used as conditioned medium for medium changes .

Effect of Lectin on Myotube Formation
Lectin wasadded directly as a concentrated solution in a small volume (0 .25 ml) of PBS to standard cultures or to cultures that previously had been grown in low [Ca"] medium to yield a final desired concentration in the medium . Myotube formation was determined by light microscopy after the cultures had been fixed with cold 5% TCA and stained with 0.25% azure B in 0.05 M phosphate buffer at pH 6.5 . Myotubes were defined as cells containing three or more nuclei in which thecytoplasmic staining in the adjacent cytoplasm was homogeneous in intensity and distribution . The total number of nuclei was determined in both mononucleated and multinucleated cells, and the number of nuclei in myotubes was expressed as a percentage of the total nuclei in the field. Because the total cell number was obtained, the detrimental effects of the experimental conditions on cell viability and proliferation could be assessed . Twelve random fields per plate were counted to represent the culture dish . This area of 1.9 mm' per plate was determined in independent studies to accurately represent values obtained from assessing larger areas of the plate. 400-1,000 nuclei were counted perexperimental point in duplicate or triplicate samples. Each experiment was done two or three times.

RESULTS
Lectin was purified by homogenization and centrifugation to obtain a postmicrosomal supernate that yielded purified lectin by a single passage through the aminoethyl Bio-gel P-150 coupled with lactose ( Fig . 1) . Before it was applied to the column, the supemate had a specific activity of 100. Passage through the affinity column and subsequent elution with PBS showed by hemagglutination that the active component was retained by the column . After the column had been rinsed with PBS (four times the sample buffer), elution with DES plus 0.3 mM lactose released a bound protein that appeared in the first few volumes after the void volume. This material had a specific activity of 110,000, yielding >1,000-fold purification .
Characterization of the purified protein included a determination of its purity by SDS-PAGE and of its molecular weight by Sephadex chromatography. Analysis of the lactose-eluted fractions with the highest specific activity obtained from the affinity column produced a single, discrete, Coomassie Blue-stained band positioned between the markers cytochrome c and myoglobin. It had an estimated subunit molecular weight of 15,000 . SDS-PAGE of the crude extract produced many stained bands, but, importantly, one of 15,000 daltons that comigrated with the purified lectin was present. After Sephadex G-75, a 15,000dalton subunit and a larger dimer fraction at 30,000 daltons were shown by hemagglutination activity .
Crude embryonic muscle extract in PBS, with a specific activity of 100, was applied to muscle cultures in conditioned medium as single and as multiple daily treatments (Fig. 2) . Application of the extraction buffer and changes of the medium had no noticeable effect on myotube formation. A single treatment with crude extract (2 .6 mg protein/ml; titer' of 256) at the onset of culture partially inhibited myotube formation on days 4 and 5 of culture, but the extent of myotube formation on day 6 was equivalent to that of untreated cultures . Sequential daily treatments with crude extract partially inhibited myotube formation during the entire culture period . Cell numbers at all points tested were equal in untreated and treated cultures . A similar experiment was performed with a crude extract of day-5 cultured The effect of the 100,000-g supernate from 14-16-d embryonic muscle dialyzed against DES plus DTT without lactose followed by PBS plus DTT on myotube formation at various times in culture. The crude extract with a hemagglutination titer' of 16 was added to the supplemented L-15 medium . O, Control untreated cultures . ", Culture receiving a single addition on day 1 of incubation. A, Culture receiving daily additions of lectin with fresh conditioned medium appropriate for the time in culture. muscle (Fig. 3) . Inhibition of myotube formation was minimal after a single addition of crude extract and was most pronounced after multiple daily additions (the effect was assessed on days 3 and 4) . No effect was found on days 5 and 6.
Purified lectin obtained from the affinity column was tested for its effect on myotube formation to show a relationship between the amount of inhibition and the concentration of the lectin applied. In early experiments, the purified lectin was added to cultures in supplemented conditioned medium . Under these conditions, in contrast to the crude extract, purified lectin had no effect on myotube formation. Consequently, conditions were modified . Purified lectin was diluted with PBS instead of conditioned medium before it was added to cultures containing supplemented medium . Muscle cultures were initially grown with decreased (Ca"]. To test its effect on the inhibition of fusion calcium was added without lectin or with increasing concentrations of lectin to promote myoblast fusion . Control cultures containing the normal [Ca +21 showed maximal myotube formation on day 3 of culture. At this time, calcium deprivation resulted in essentially no myotube formation . Fig. 4 shows that, in the absence of lectin, elevation of [Ca+ 2] resulted in myotube formation in the ensuing 8 h. Lectin with a specific activity of 110,000 added at a concentration of 0.0225 pg/ ml (titer -' of 2.5) had no effect on myotube formation . Higher concentrations of lectin, i.e ., 0.045 1íg/ml (titer-' of 5.0) to 0.0901íg/ml (titer' of 10), inhibited myotube formation 50 to 90%, 6 h after lectin addition . 8 h after lectin was added, its effect diminished, and the amount of myotube formation approached that of untreated cultures . Addition of 0.3 M lactose with the lectin cancelled its effect .
That lectin did not effect myotube formation when it was diluted in supplemented medium suggested that inactivation was occurring that could account for the temporary inhibition of myotube formation observed in the experiments described above. We tested for temperature lability and possible inactivation by components in the supplemented medium by hemagglutination assays . On the basis of these results, we applied lectin purified by affinity chromatography in PBS with L-15 solution (1 .5 mM Ca") to calcium-deprived cultures with and without HS and CEE to test for maximal effects of the lectin on myotube formation (Fig. 5) . We minimized potential heat inactivation in the absence of HS and CEE by replenishing with fresh lectin. Fusion occurred normally and on schedule in lectin-free control cultures with HS and CEE after [Ca +21 was increased. Fig. 6A and B depicts myotubes formed under these conditions. Deletion of HS and CEE produced slightly reduced myotube formation (Fig . 5) . Fig. 6 C and D shows that the cells have become more elongate and that the amount of fusion is diminished but still quite evident. Although the myotubes are narrow, they still meet our criteria for the definition of myotubes. Cultures treated singly with lectin in the absence of HS and CEE showed partial inhibition of myotube formation up to 12 h after [Ca +21 was elevated, but myotube formation approximated control cultures by 14 h. Cultures in which the lectin was replenished every 2 h in the presence of 1 .5 mM Ca" showed myotube formation below 5% up to 12 h, with a slight increase thereafter . Fig. 6 E shows no myotubes  Fig. 6 F shows the typical relationship of cells passing over one another or contacting one another but evidently not with continuous cytoplasm.
A lectin extracted from 14-16-d pectoral embryonic chicken muscle with DES and lactose (19) was purified using Bio-Gel P-150 coupled with lactose by reductive amination (1). This procedure usually produced about 1 mg of purified lectin from 20 g wet wt of embryonic muscle . Affinity chromatography was used similarly by Den and Malinzak (4) with an asialofetuin Sepharose column and by Nowak et al. (20) with a Sepharose 4 B coupled to a-aminophenyl-/3-lactoside . Lectins isolated by these methods showed a subunit molecular weight of 15,000 on SDS-PAGE and a dimeric molecular weight of 30,000 by Sephadex G-100 gel filtration (4,20). Furthermore, Nowak et al . (20) showed that the lectin had an isoelectric THE JOURNAL OF CELL BIOLOGY " VOLUME 85, 1980 point of 4.0 . All of the purified proteins, including the ones described in this report, had hemagglutinating activity that was inhibited by lactose (4,20). By these criteria, the proteins have similar identities and probably are similar to those reported by Teichberg et al . (27) and by Gartner and Podleski (7). Isolation of these lectins from muscle tissue or muscle cultures of salt-sugar extraction probably involves both cytoplasmic and cell surface material. Lectin activity was reported in the particulate and soluble fractions from L 6 skeletal muscle culture homogenates (7). Immunochemistry showed that the lectin existed in cultured skeletal muscle myoblasts and myotubes both as constituents of the cell surface and diffusely spread throughout the cytoplasm (20) . Although such information does not exist for embryonic muscle tissue, it is presumed that a similar distribution exists .
The presence of high concentrations of the endogenous lectin during the myoblast fusion phase in both muscle tissue and cultures suggests that it is a developmentally regulated protein, perhaps involved in the recognition and fusion process (7,19). Den and co-workers (4, 6) concluded that there is insufficient evidence to warrant such conclusions . They treated chicken muscle cultures with various galactose sugars but were unable to inhibit myotube formation, i.e ., the end product of cell recognition, adhesion, and fusion . In contrast, inhibition of myotube formation was obtained for rat muscle cell lines (4,7) . This species and/or cell line difference in response has not been resolved . Our unpublished observations show that continuous treatment of chicken muscle cultures with lactose results in retardation of myotube formation by 48 h, but the inhibition is overcome, with the final extent of fusion equivalent to untreated cultures. Cultures were treated continuously from 24 h after plating with four sugars added directly to the culture medium at 0.15 M. At 72 h, a-methyl-D-mannoside, a-methyl-D-glucoside, and a-L(-)fucose did not significantly affect fusion, as determined by visual inspection. Lactose-treated cultures contained little myotube formation; cell division was not impaired, as evidenced by the confluent cell layer. At 96 h, all experimental and control cultures were comparable with regard to the extent of myotube formation, suggesting that lactose had transiently inhibited fusion .
In this study, we show that myotube formation is reproducibly inhibited by added 15,000-dalton added to the supplemented medium in the purified form than as a component of a crude extract, which suggests that secondary components may be associated with the lectin that serve as protective agents . It is assumed that added lectin reacts with the cell surface, resulting in incomplete incorporation and thereby leaving the lectin accessible for rapid inactivation .
Plant lectins have been shown in similar studies to interfere with myotube formation. Den et al . (5) tested the effects of concanavalin A (con A), wheat germ agglutinin, soybean agglutinin, abrin, and Lens culinaris lectin on cultures that had been calcium deprived . They found myotube formation to be inhibited 0-60% . On the basis of these observations, they suggested that the effects were the result of either binding to specific glycoproteins essential for fusion or nonspecific steric hinderance of cell contact necessary for fusion . Sandra et al . (21,22) studied more closely the effect of con A and showed that this lectin would inhibit myotube formation under standard culture conditions, provided the cells were mildly pretrypsinized before lectin addition . This trypsin-lectin treatment resulted in long lasting effects . It produced 60% inhibition of myotube formation without crosslinking and 80% inhibition with con A and glycogen. Cell surface replicas show that normal myotube formation coincided with the dispersed state of con A binding sites on myoblast cell surfaces . Inhibition of myotube formation correlated closely with a clustered state of the con A surface receptors . On the basis of these data, they suggested that con A binding affected the membrane fluidity by cross-linking, and that it is unclear whether cell surface glycoproteins are involved in fusion, are cross-linked, or are adventitiously trapped . The effect was due more to physical interference of the 62 4 THE JOURNAL OF CELL BIOLOGY " VOLUME 85, 1980 surface than to actual involvement of fusion sites . Unfused myoblasts continued to make contact at both their end processes and their lateral surfaces. The lactose-binding lectin isolated from embryonic muscle is more effective than plant lectins in inhibiting myotube formation. The 15,000-dalton muscle lectin yielded >90% inhibition at picomole concentrations, suggesting that it has higher specificity and a greater potential for physiological effects than the plant lectins that yielded effects at millimole concentrations . Such differences may result from varied experimental conditions . In current studies, cultures are handled in the same way as that described for Fig. 5 and are treated with 15,000-dalton muscle lectin (0 .09 Ng/ml), con A, or peanut lectin (0 .09-125 Fig/ml), either as single additions without a medium change or as multiple additions with medium changes. Initial results show a decrease in myotube formation with the muscle lectin comparable to that depicted in Fig . 5 . In contrast, the plant lectins did not decrease myotube formation.
We propose that the endogenous lactose-binding lectin has a natural role in muscle development . Our studies show that it does not affect cell proliferation and cell motility (unpublished timelapse studies), and that in this way it is comparable to con A (22) . In untreated skeletal muscle cultures, myoblasts align before fusion in such a way that contacts are made at the ends and lateral surfaces of the cells. In cultures in which [Ca +2] is decreased to inhibit myotube formation, our studies and those of others (11) show that cell contact occurs by an elongate microspike . These initial sites of contact may result in gap junction formation, as has been shown in closely contacted myoblasts (12) and in fusing myoblast membrane preparations (3) . These junctions could establish a "communication" between the cells, promoting the modification of the cell surfaces for fusion perhaps, as suggested by Kalderon and Gilula (13), via the incorporation of intracellular vesicles within the cell membrane to produce phospholipid-enriched domains free of intramembrane particles . However, regions of the cell membrane containing gap junctions would not be the sites of membrane dissolution and eventual cytoplasmic continuity between adjacent cells . Such continuity can only be achieved if the adjacent myoblasts are closely apposed . We propose that the endogenous lactose-binding lectin is involved in the close apposition of myoblasts .
We interpret the results of our studies in the following way . Once the initial cell contact and communication is accomplished between myoblasts, a prolonged cell-cell contact is necessary to allow fusion of the respective membranes . Continued cell-cell apposition, promoted by increasing the [Ca +21, involves the lactose-binding lectin .
When present in sufficient quantity in the cell membrane, it binds with specific lactose-containing receptors on adjacent cells and thus acts to hold the cells together. However, if exogenous lactose-binding lectin is present in active form, the adhesive sites are experimentally covered, as indicated by the minimal amount of lateral cell surface apposition observed in our studies. Inactivation of the added lectin by heat or components of HS and CEE leads to continued myotube formation . However, it appears that this occurs by the cytoplasm's becoming confluent at the initial sites of cell contact near the microspikes inasmuch as the myotubes formed are initially long and thin . This suggests that myoblast fusion can occur in discrete regions and that the added lectin is capable of temporarily inhibiting membrane apposition at these sites . One would assume that the proposed role for the endogenous lactose-binding lectin in developing skeletal muscle requires specificity . That similar Lectins are present in a variety of tissues (15) speaks against such specificity . However, we suggest that too little information currently exists to conclude that they are identical . It is not unreasonable to surmise that portions of these Lectins are similar and thus provide for similar means of isolation and common antigenic determinants . The variable regions of the molecules would provide for specificity of function and, thus, for variations in distribution within tissues .