0022-1554/92/$3.30
Vol. 40, NO. 10, pp. 1511-1516, 1992 Printed in USA.
The Journal of Histochemistry and Cytochemisuy Copyright 0 1992 by The Histochemical Society, Inc.
Original Article
I Mosaic Lectin and Enzyme Staining Patterns in Rat Skeletal Muscle SVEND KIRKEBY,' THORKILD C. B@G-HANSEN, and DENNIS MOE Department of Oral Function (SK), Protein Laboratory (TCB-H), and Department of Microbioloa (DM), Hedtb Science Faculty, Universzty of Copenbagen, Copenhagen, Denmark. Received for publication December 27. 1991 and in revised form April 21, 1992; accepted May 4, 1992 (1A2548).
We compared the localizations of lectin binding and activity for myosin ATPase and succinic dehydrogenase in sections of the gracilis, soleus, and masseter muscles from 10and 60-day-old rats. In the 60-day-old rats, incubation of the muscle sectionswith the lectins ConA, GS-11, HPA, and jacalin gave rise to a mosaic staining pattern, especially in the gracilis muscle, in which the same fibers were strongly stained for ConA, GS-11, and IPA, whereas the staining with jacalin in these fibers was weak, and vice versa. There w a s no correspondencein the staining patterns for the enzymes and the lectins. In the masseter muscle only GS-11gave rise to distinctdifFerencesin the staining intensity betweenmuscle fibers. In 10-day-oldrats all fibers in the muscles were moderately stained with C o d , HPA, and jacalin, whereas a chess-
Introduction Complex glycoconjugates are of importance for a number of biological events such as interaction between cells, development and growth, function of cells, and changes in function. Lectin histochemistry is a major analytical tool for investigation of cell glycoconjugates, since the location of specific lectin binding compounds can be determined in situ. Lectin binding in skeletal muscle can be shown in both the sarcoplasm and sarcolemma, and lectin binding in skeletal muscle has been used to describe carbohydrate constituents of the sarcolemma in normal muscle and membrane changes in various neuromuscular diseases (Moggio et al., 1989; Voit et al., 1989; Capaldi et al., 1985). The specific location and function of the sarcolemmal glycoconjugates are not known, and lectins have been used only occasionally to characterize individual muscles. For this purpose, enzyme histochemical demonstration of activity for myosin ATPase and/or succinic dehydrogenase (SDH) is the method of choice. To determine if there is a relationship between the localization of enzyme activity and the specificityof the sarcoplasmic glycoconjugates, we
Correspondence to: Svend Kirkeby, Dept. of Oral Function, Dental School, Health Science Faculty, University of Copenhagen, N)rre AllC 20, DK 2200, Copenhagen, Denmark.
board staining pattern could be observed after incubation with GS-II. In an extract of hindleg muscle from 60-day-old rats there was strong affinity for ConA and HPA and weak a f f i t y for GS-IIand jacalin, as shown by dot-blotting. After electrophoresis and blotting to n i d u l o s e membranes, three muscle protein bands with apparent molecular weights of 100,000, 90,000, and 43,000 showed affinity for ConA, HPA, and GS-II, whereas no bands were jacalin positive. The complex lectin staining pattern in skeletal muscle might be related to development, specialization, and function of the muscles. ( J Hstochem Cytochem 40:15f 1-1516, 1992) KEY WORDS: Skeletal muscle; Development; Lectins; Myosin ATPase; Succinic dehydrogenase.
have compared the staining pattern of ATPase and SDH with the distribution of binding of 16 different lectins in rat skeletal muscle. The lectin staining patterns in muscles from 10-day-and 60day-old rats were compared to identify a possible relationship between lectin binding and the maturation of the muscles. Finally, the carbohydrate content in a muscle extract was characterized by lectins on dot-blots and after electrophoresis.
Materials and Methods Six male Wistar rats weighing 270-300 g (about 60 days old) and four male rats weighing 20 g (about 10 days old) were sacrificed by cervical dislocation. The masseter, gracilis, and soleus muscles were removed. For the histochemicalstudies, specimens were frozen in kopentanc cooled to - 150°C with liquid nitrogen and cut on a cryostat in 6-wm serial sections. Muscle extracts for dot-blots and electrophoresis were obtained from mixed leg muscles of the 60-dayrats. Two grams of muscle were homogenized in 10 ml 0.1 M Tiis buffer. pH 7.2, with 40 mM 3-[(3-~holamidopropyI)dimethylammoniol-1-propanesulfonate (CHAPS) and 1 mM phenylmethylsulfonylfluoride (PMSF). The homogenization was performed with a Waring blcndor for 30 sec at 4%. The homogenate was stored at 4'C for 15 min and thereafter centrifuged at 27,000 x g for 30 min. The supernatant was used for the analyses.
Dot-blots. The muscle supernatant was diluted four times with Tiis buffer and 1 pl of this solution was applied to nitrocellulose paper (mem-
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brane filter 0.2 pm; Schleicher & Schuell, Dassel, Germany). Before incubation with Iectins, excess binding sites were blocked by incubation for 4 min at 20’C in TBS containing 4% Twccn-20,followed by two washes in TBS.
ticed after incubation with AAA, LTA, PSA, LCA, WGA, PHA-E, and PHA-L. The following lectins gave rise to a weak or nondetectable sarcoplasmic reaction: UEA-I, PNA, GS-I, DBA, and
Electrophoresis and Western Blotting. SDS electrophoresis was carried out with a Pharmacia PhastSystem (Uppsala, Sweden) on a 12.5% homogeneous PhastGel with SDS buffer strips. Each lane was loaded with 2 pI of the supematant prepared with the SDS-containingbuffer. The elecuophoresis was performed for 65 accumulated volt hours at a current of 10 mA. Electrotransfer to nitrocellulose was performed with Phast Transfer Semidry Electrophoretic Xansfer System (Pharmacia). The transfer buffer was a 0.05 M Xis-glycine buffer, pH 8.3, containing 20% methanol. The transfer was performed at 20 V with current 25 mA. The transfer was checked by staining with acid Fast Green FCF (1% in 10% acetic acid). Before incubation with lectins, excess binding sites were blocked as above.
SBA.
Le&. The following biotinylated lectins (Kem-En-Tec;Copenhagen, Denmark) were used in this study: L-fucoseagglutinins: UfexeumpaeusI(UEA-1);Aleuria aurantrb (AAA); Lotus tetragonofobus (LTA). Mannose andlor glucose agglutinins: Concanavalin A (ConA);Pisum sativum (PSA); Lens cufinaris (LCA). Galactose agglutinins:Peanut (PNA); Griffonia simpficifofiaI (GS-I); Astocarpus integrrjcofia (jacalin). N-acetyl-a-D-dactosaminyl agglutinins: Heficpomatia (HPA); Dofichos &$’orus (DBA); Glycine max (SBA). N-aceryl-fl-D-glucosaminylagglutinins: Triticum vulgaris (WGA); Griffonia simpficzyofia II (GS-11). Oligosaccharide agglutinins: Phaseofus vulgaris erythroagglutinin (PHA-E); Phaseofus vulgaris leucoagglutinin (PHA-L). Stainingof Sections. The lectin staining of the sections was performed as described in a previous report, using unfixed sections and avidin-alkaline phosphatase to visualize the binding sites of the biotinylated lectins (Kirkeby et al., 1991). Localization of activity for myosin ATPase was performed according to Round et al. (1981) and activity for SDH was shown according to Kitkeby et al. (1988). Staining for Lectin Binding on Nitrocellulose Paper. After washing with TBS the blots were: (a) incubated for 24 hr at 4’C with biotinylated lectins diluted to a concentration of 10 pg/ml with TBS containing 1 mM CaCIz, MnC12, MgC12 and 0.05% Tween-20; (b) rinsed twice for 5 min in TBS buffer; (c) incubated for 30 min at room temperature with alkaline phosphatase-conjugatedavidin (Dakopatts 365; Glomp, Denmark) diluted 1:600with TBS; (d) rinsed three times for 5 min in TBS with 2.5 mg levamisole (Sigma L 9756; St Louis, MO) per 10 ml; (e) incubated for 15 min at room temperature in 10 ml Tris buffer (pH 9.5, 0.1 M), 10 mM MgCIz, 2.5 mg 5-bromo-4-chloroindoxyl phosphate (Bachem M-1780; Torrance, CA) in 0.1 ml N,N-dimethylformamide,3 mg nitroblue tetrazolium (Sigma N 6876), and 2.5 mg levamisole. For controls, 2 pl of the Xis buffer were applied to the nitrocellulose paper incubated with the different lectins and stained for possible lectin binding. In other experiments the myosin preparation and the muscle sections were incubated in buffer without lectin and stained for possible nonspecific avidin-biotin-alkaline phosphatase reaction.
Results The staining pattern after lectin incubation was very complex in muscle sections from the 60-day rats. Reaction could be recorded in different sites such as sarcoplasm, sarcolemma, connective tissue, and capillaries. A strong or moderate sarcoplasmic reaction, with the same staining in all fibers in the three muscles, was no-
Incubation with ConA, GS-11, HPA, and jacalin resulted in a mosaic staining pattern, with differences in the lectin staining intensity among individual fibers. The reaction was present as discrete granules and as diffuse cytoplasmic staining. Of the muscle investigated, the mosaic staining pattern was most apparent in the gracilis muscle. In this muscle a fiber was stained with the same intensity (strong or weak) after incubation with ConA, HPA, and GS-11, whereas incubation with jacalin resulted in a reversed reaction in most fibers. Thus, by comparing serial sections, a fiber that showed high affinity to ConA, HPA, and GS-I1 was weakly stained with jacalin, and fibers that were jacalin positive were weakly stained with the three other lectins (Figures la-Id). The muscle contained both Type I fibers (low activity for ATPase after incubation at pH 9.4) and Type I1 fibers (high activity for ATPase after incubation at p H 9.4) (Figure le). Incubation for SDH showed that both “white” fibers (low SDH activity) and “red” fibers (high SDH activity) were present (Figure lf). Serial sections that were incubated to show activity for lectins, ATPase, and SDH revealed that there was no relation between the lectin staining pattern and the intensity of enzyme activity in the fibers (Figures la-If). In the soleus muscle the staining reactions in the fibers after incubation with the different lectins was more homogeneous, with many fibers showing a moderate intensity. However, it was obvious also in this muscle that most of the fibers that were strongly ConA, GS-11, and HPA positive were jacalin negative, and vice versa (Figures 2a-2d). Again, there was no relation between the reactions for the lectins and the activity of myosin ATPase. In the superficial part of the masseter muscle, most fibers were stained moderately after incubation with ConA, GS-11, HPA, and jacalin. In the deep part of this muscle there appeared to be a more diverse reaction. In particular, incubation with GS-I1 resulted in a differentiated staining pattern, with some fibers reacting weakly and other fibers showing moderate or strong staining (Figure 2e). In contrast, the majority of the fibers were moderately stained with ConA (Figure 2f) and moderately to weakly with HPA and jacalin. All fibers were strongly ATPase positive. In the 10-day-oldrats, incubation with GS-I1resulted in strongly, moderately, and weakly stained muscle fibers (Figure 3a), while there was moderate staining in all the fibers of the three muscles examined after incubation with HPA, jacalin, and ConA (Figures 3b-3d). This means that in these young rats a lectin mosaic staining pattern could only be observed after incubation with GS-11. The lectin staining was mostly present as coarse granules, often as a part of a network as shown with ConA (Figure 3e). In the gracilis and soleus muscles a few fibers showed weak activity for myosin ATPase (Type I), whereas all the other fibers displayed a strong enzyme reaction (Type 11) (Figure 3f). In the control sections incubated without lectins there was very weak cytoplasmic staining. When dot-blots of the muscle extracts were incubated with lectins, ConA and HPA gave rise to a strong reaction, while weak staining appeared after incubation with GS-I1 and jacalin (Figure 4). In the blot incubated i n buffer without lectin and thereafter incubated with avidin-phosphatase, there was very weak staining for alkaline phosphatase (Figure 4).
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Electrophoretically separated muscle extracts were blotted to nitrocellulose paper and incubated with ConA, GS-11, HPA, and jacalin. Lectin-stained bands were obtained after incubation with ConA, GS-I1 and HPA, whereas no bands were present after incubation with jacalin (Figure 5 ) . The molecular weights of the ConAstained bands ranged from 120,000 to 43,000, while the bands stained with GS-I1 and HPA ranged from 100,000 to 43,000. Although many bands were HPA and ConA positive, only three GSI1 bands were clearly expressed. The apparent molecular weights of the GS-I1 bands were 100,000,90,000,and 43,000. These bands appeared also to be ConA and HPA positive. No nonspecific bands were observed when electrophoretic blots were incubated for reaction with avidin-alkaline phosphatase.
Discussion In the muscle sections a number of lectins detected sarcoplasmic
carbohydrate constituents. The exact localizations of the intracellular lectin binding sites are not known but in normal muscles most if not all of these glycoconjugates are membrane bound or are part of the myofibrillar proteins, since a membrane-deprived high-speed muscle supernatant contains virtually no soluble lectin binding glycoproteins (Kirkeby et al., 1992). Four of the lectins tested resulted in a sarcoplasmic mosaic staining pattern in the sections from adult rat muscle. These were ConA, HPA, GS-11, and jacalin. It is interesting that all four lectins have different sugar preferences. ConA, PSA, and LCA were the three mannoselglucose agglutinins tested in this study. Whereas incubation with ConA resulted in a mosaic staining in the sections of the gracilis, and partly in the soleus, LCA and PSA stained all fibers with the same intensity in these muscles. That PSA and LCA detect fibers that are not stained with ConA could be due to the broader pattern of sugar agglutination of the two former lectins. In contrast to ConA, LCA
Figure 1. (a-d) Lectin staining and (0-1) enzyme activity in gracilis muscle from 60-dayold rat. (a) ConA; (b) HPA; (c) GS-11; (d) jacalin. A mosaic staining pattern in the muscle section is evident. The same fibers that react strongly after incubation with ConA, HPA, and GS-II are weakly stained after incubation with jacalin and vice versa. (e) Myosin ATPase: (1) SDH. a-1 are serial sections. Note that there is no connection between lectin staining and enzyme reaction in the muscle fibers. Original magnification x 120. Bar = 50 pm.
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Figure 2. Lectin staining in (a-d) soleus muscle and (e,f) masseter muscle from 60day-old rat. (a) ConA; (b)GS-II; (c) HPA; (d) jacalin. In the soleus muscle the mosaic staining pattern for the lectins is less evident than in the gracilis muscle. (e) GS-II; (f) ConA. The mosaic staining for the lectins in the masseter muscle is evident only after incubationwith GS-II. Original magnification x 120. Bar = 50 wm.
and PSA might also detect N-acetylglucosamine (provided a fucose residue is present in the C-6 position) (Debray et al.. 1983). GS-I1 is an N-acetyl-fi-D-ghcosaminyl agglutinin. In a previous report we described a concordance between the staining intensity for GS-I1 and ConA and the PAS reaction in fibers from the rat gracilis muscle (Kirkeby et al., 1991). It was suggested that GSI1 detects muscle glycogen or glycoproteins attached to glycogen and ConA detects the same but also unidentified sarcoplasmic glycoproteins. Among the GalNAc-specificlectins (HPA, DBA, and SBA), only incubation with HPA gave rise to a sarcoplasmic mosaic staining pattern. Furthermore, HPA also detected the sarcolemma, whereas incubation with DBA and SBA resulted only in very weak, homogeneously disturbed sarcoplasmic reaction. The specificity of SBA is high for GalNAcal+3Gal (human blood group A). DBA detects A determinant and the Forssman-active pentasaccharide
(GalNAca- GalNAcP- 3Galal+4Galfil+4Glr,. while HPA agglutinates A, F, and Tn determinants [GalNAcal+O to Ser(Thr) of the protein core] (Wu et al., 1988). The Galfi+ specific lectin jacalin also gave rise to a mosaic staining pattern in the muscle sections, but the distribution tended to be the reverse of that obtained after HPA incubation. The other Gal-specific lectins, PNA and GS-I, did not detect sarcoplasmic constituents. Both PNA and jacalin have high specificity and af,finity for GalPl+3GalNAc, which forms the core disaccharide of 0-linked oligosaccharides (T antigen) (Wu et al.. 1988). The mosaic lectin staining pattern was not equally expressed in the three muscles investigated. It was evident in the gracilis, less distinct in the soleus, and in the masseter it was evident only for GS-11. These differences in the lectin staining pattern might be correlated with differences in muscle function. Gracilis is fast twitch, soleus is slow twitch, and masseter is a masticatory muscle which +
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LECTIN AND ENZYME STAINING OF SKELETAL MUSCLE
Figure 3. ( a d ) Lectin staining and (f) ATPase activity in gracilis muscle from 10-dayold rats. (a) GS-II; (b) HPA; (c) jacalin; (d) ConA. There is variation in the staining intensity after incubation with GS-11, while all fibers react moderatelyafter incubationwith ConA. HPA, and jacalin. (e) A higher magnification of ConA staining in the muscle fibers. Note the network-likestaining pattern. (1) ATPase reaction in the muscle fibers. Most fibers are strongly stained, but a few fibers are ATPase negative. Original m a g nifications: a-d,f x 204; e x 985. Ears: ad,f = 20 pm; e = 5 pm.
both functionally and enzymatically (myosin ATPase) is different from limb muscles. In the 10-day-old rats no mosaic lectin staining pattern was observed in any of the muscles investigated after incubation with ConA, HPA, or jacalin. At this age the differentiation of the muscle fibers into the mature, specific types had barely started, since almost all the fibers were equally strongly stained after incubation for myo-
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A
B
i
C
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E
Figure 4. Dot-blotof the muscle extract from hindleg muscles stained for lectin binding. (A) ConA; (e)GS-II; (C) HPA; (0)jacalin; (E) blank (incubated in buffer without lectin).
sin ATPase. At this stage of muscle specialization the muscles can all be described as slow. Furthermore, after lectin incubation the colored sarcoplasmic reaction product in the muscle fibers from 60-day-old rats was present as discrete granules and as a diffuse cytoplasmic reaction. In the very young rats the sarcoplasmic reaction consisted mainly of coarse granules, often as part of a network. Therefore, the lectin-staining patterns in very young and in more mature rat muscles are different, with a much more complex pattem in older muscles indicating a connection between muscle maturation and the presence of sarcoplasmic lectin binding sites. Using a fucose-specific lectin, Faze1 et al. (1989) have shown that in the embryonic heart the expression of the fucosylated glycoconjugates is stage-dependent and is therefore probably genetically regulated. There was no concordance between fiber reactions for enzyme activity and lectin staining in the adult muscles. This is in contrast to Ymagami et al. (1985), who described a correlation between staining for myosin ATPase and ConA. However, their material was
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=BY,
In conclusion, the lectin-staining pattern in skeletal muscle is very complex. It might be related to development, specialization, and function of the individual muscles. There is no relationship between the mosaic lectin staining pattern and that obtained after incubation for myosin ATF’ase or succinic dehydrogenase.
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Literature Cited
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Capaldi J. Dunn MJ, Scwry CA. Dubowitz V (1985): Lectin binding in human skeletal muscle: a comparison of 15 different kctins. Histochcm J 1791 Debray H. Pierce-Creta1 A. Spik G, Montrevil J (1983): Affinity of ten insolubilized lectins towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides. In Beg-Hanscn TC, Spcnglcr G, eds. Lectins. biology, biochemistry. clinical biochemistry. Vol3. Berlin. de Gruytcr, 335 Faze1 AR. Sumida H. Schulte BA. Thompson RP (1989): Lectin histochcmistry of the embryonic heart: fucose-specific lectin binding sites in dcveloping rats and chicks. Am J Anat 184:76
Figure 5. Lectin staining of electrophoreticallyseparated and blotted muscle proteins. (A) ConA; (E) GS-II; (C) HPA (D) jacalin; (E) Fast Green; (F)molecular weight of marker proteins (in kilodaltons) stained by Fast Green.
Kirkcby S, Bag-Hanscn TC. Moe D. Garbarsch C (1991): Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures. Histochem J 23:345
4
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B
C
D
E
43
F
post-fixed in Carnoy’ssolution and pre-treated with trypsin before the incubation with lectins. It is likely that with this treatment other carbohydrate structures are exposed for reaction with ConA than those accessible in fresh untreated muscle, as in our material. ConA, HPA. and GS-I1stained sarcoplasmic components in the same fibers in leg muscles. In a blot of electrophoretically separated muscle proteins, three protein bands appeared to be detected by ConA, HPA, and GS-11. It might be suggested that these electrophoretically demonstrable lectin-positive proteins are at least partly responsible for the histochemically demonstrable lectin reaction. In addition, a number of protein bands were stained with ConA and HPA. and a band with apparent molecular weight of 120,000 seems to be specific for ConA binding. No bands were showed to have jacalin affinity, either because jacalin-positive glycoproteins are not present in the blot after electrophoresis or because the glycoconjugates positive for jacalin are glycolipids unable to migrate in the polyacrylamide gel.
Kirkcby S. Garbanch C, Matthiessen ME, Beg-Hansen TC.Moe D (1992): Changes of soluble glycoproteins in dystrophic (dy/dy)mouse muscle shown by lectin binding. Pathobiology. in press Kirkeby S. Moe D. Vilmann H (1988): Esterase profile of human masseter muscle. J Anat 157:79 Moggio M. Jann S. Adobbati L, Prcllc A. Gallanti A. Fagiolari G, Pellegrini G, Scarlato G (1989): Ultrastructural localization of calcium binding sites on human muscle cell surface. Muscle Nerve 12:910 Round JM, Matthcws Y, Jones DA (1981): A quick, simple and reliable histochemical method for ATPase in human muscle preparations. Histochcm J 12:707 Voit T, Patcl K. Sewry CA, Strong PN. Dubowitz V, Dunn MJ (1989): Membran-verandrungen bei DuchennelBecker-Muskeldystrophie:Lectinbindung und Dystrophin-lokalisation.Monatsschr Kinderheilkd 137:20 Wu AM, Sugii S. Herp A (1988): A guide for carbohydrate specificities of lectins. In Wu AM, cd. The molecular immunology of complex carbohydrates. Ncw York, London, Plenum Press. 819
Yamagami T, Hosaka M. Mori M (1985): Classification of skeletal muscle fibres by comparison of enzyme histochemistry with lectin binding. Cell Mol Biol 31:241
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Vol. 40, NO. 10, pp. 1511-1516, 1992 Printed in USA.
The Journal of Histochemistry and Cytochemisuy Copyright 0 1992 by The Histochemical Society, Inc.
Original Article
I Mosaic Lectin and Enzyme Staining Patterns in Rat Skeletal Muscle SVEND KIRKEBY,' THORKILD C. B@G-HANSEN, and DENNIS MOE Department of Oral Function (SK), Protein Laboratory (TCB-H), and Department of Microbioloa (DM), Hedtb Science Faculty, Universzty of Copenbagen, Copenhagen, Denmark. Received for publication December 27. 1991 and in revised form April 21, 1992; accepted May 4, 1992 (1A2548).
We compared the localizations of lectin binding and activity for myosin ATPase and succinic dehydrogenase in sections of the gracilis, soleus, and masseter muscles from 10and 60-day-old rats. In the 60-day-old rats, incubation of the muscle sectionswith the lectins ConA, GS-11, HPA, and jacalin gave rise to a mosaic staining pattern, especially in the gracilis muscle, in which the same fibers were strongly stained for ConA, GS-11, and IPA, whereas the staining with jacalin in these fibers was weak, and vice versa. There w a s no correspondencein the staining patterns for the enzymes and the lectins. In the masseter muscle only GS-11gave rise to distinctdifFerencesin the staining intensity betweenmuscle fibers. In 10-day-oldrats all fibers in the muscles were moderately stained with C o d , HPA, and jacalin, whereas a chess-
Introduction Complex glycoconjugates are of importance for a number of biological events such as interaction between cells, development and growth, function of cells, and changes in function. Lectin histochemistry is a major analytical tool for investigation of cell glycoconjugates, since the location of specific lectin binding compounds can be determined in situ. Lectin binding in skeletal muscle can be shown in both the sarcoplasm and sarcolemma, and lectin binding in skeletal muscle has been used to describe carbohydrate constituents of the sarcolemma in normal muscle and membrane changes in various neuromuscular diseases (Moggio et al., 1989; Voit et al., 1989; Capaldi et al., 1985). The specific location and function of the sarcolemmal glycoconjugates are not known, and lectins have been used only occasionally to characterize individual muscles. For this purpose, enzyme histochemical demonstration of activity for myosin ATPase and/or succinic dehydrogenase (SDH) is the method of choice. To determine if there is a relationship between the localization of enzyme activity and the specificityof the sarcoplasmic glycoconjugates, we
Correspondence to: Svend Kirkeby, Dept. of Oral Function, Dental School, Health Science Faculty, University of Copenhagen, N)rre AllC 20, DK 2200, Copenhagen, Denmark.
board staining pattern could be observed after incubation with GS-II. In an extract of hindleg muscle from 60-day-old rats there was strong affinity for ConA and HPA and weak a f f i t y for GS-IIand jacalin, as shown by dot-blotting. After electrophoresis and blotting to n i d u l o s e membranes, three muscle protein bands with apparent molecular weights of 100,000, 90,000, and 43,000 showed affinity for ConA, HPA, and GS-II, whereas no bands were jacalin positive. The complex lectin staining pattern in skeletal muscle might be related to development, specialization, and function of the muscles. ( J Hstochem Cytochem 40:15f 1-1516, 1992) KEY WORDS: Skeletal muscle; Development; Lectins; Myosin ATPase; Succinic dehydrogenase.
have compared the staining pattern of ATPase and SDH with the distribution of binding of 16 different lectins in rat skeletal muscle. The lectin staining patterns in muscles from 10-day-and 60day-old rats were compared to identify a possible relationship between lectin binding and the maturation of the muscles. Finally, the carbohydrate content in a muscle extract was characterized by lectins on dot-blots and after electrophoresis.
Materials and Methods Six male Wistar rats weighing 270-300 g (about 60 days old) and four male rats weighing 20 g (about 10 days old) were sacrificed by cervical dislocation. The masseter, gracilis, and soleus muscles were removed. For the histochemicalstudies, specimens were frozen in kopentanc cooled to - 150°C with liquid nitrogen and cut on a cryostat in 6-wm serial sections. Muscle extracts for dot-blots and electrophoresis were obtained from mixed leg muscles of the 60-dayrats. Two grams of muscle were homogenized in 10 ml 0.1 M Tiis buffer. pH 7.2, with 40 mM 3-[(3-~holamidopropyI)dimethylammoniol-1-propanesulfonate (CHAPS) and 1 mM phenylmethylsulfonylfluoride (PMSF). The homogenization was performed with a Waring blcndor for 30 sec at 4%. The homogenate was stored at 4'C for 15 min and thereafter centrifuged at 27,000 x g for 30 min. The supernatant was used for the analyses.
Dot-blots. The muscle supernatant was diluted four times with Tiis buffer and 1 pl of this solution was applied to nitrocellulose paper (mem-
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brane filter 0.2 pm; Schleicher & Schuell, Dassel, Germany). Before incubation with Iectins, excess binding sites were blocked by incubation for 4 min at 20’C in TBS containing 4% Twccn-20,followed by two washes in TBS.
ticed after incubation with AAA, LTA, PSA, LCA, WGA, PHA-E, and PHA-L. The following lectins gave rise to a weak or nondetectable sarcoplasmic reaction: UEA-I, PNA, GS-I, DBA, and
Electrophoresis and Western Blotting. SDS electrophoresis was carried out with a Pharmacia PhastSystem (Uppsala, Sweden) on a 12.5% homogeneous PhastGel with SDS buffer strips. Each lane was loaded with 2 pI of the supematant prepared with the SDS-containingbuffer. The elecuophoresis was performed for 65 accumulated volt hours at a current of 10 mA. Electrotransfer to nitrocellulose was performed with Phast Transfer Semidry Electrophoretic Xansfer System (Pharmacia). The transfer buffer was a 0.05 M Xis-glycine buffer, pH 8.3, containing 20% methanol. The transfer was performed at 20 V with current 25 mA. The transfer was checked by staining with acid Fast Green FCF (1% in 10% acetic acid). Before incubation with lectins, excess binding sites were blocked as above.
SBA.
Le&. The following biotinylated lectins (Kem-En-Tec;Copenhagen, Denmark) were used in this study: L-fucoseagglutinins: UfexeumpaeusI(UEA-1);Aleuria aurantrb (AAA); Lotus tetragonofobus (LTA). Mannose andlor glucose agglutinins: Concanavalin A (ConA);Pisum sativum (PSA); Lens cufinaris (LCA). Galactose agglutinins:Peanut (PNA); Griffonia simpficifofiaI (GS-I); Astocarpus integrrjcofia (jacalin). N-acetyl-a-D-dactosaminyl agglutinins: Heficpomatia (HPA); Dofichos &$’orus (DBA); Glycine max (SBA). N-aceryl-fl-D-glucosaminylagglutinins: Triticum vulgaris (WGA); Griffonia simpficzyofia II (GS-11). Oligosaccharide agglutinins: Phaseofus vulgaris erythroagglutinin (PHA-E); Phaseofus vulgaris leucoagglutinin (PHA-L). Stainingof Sections. The lectin staining of the sections was performed as described in a previous report, using unfixed sections and avidin-alkaline phosphatase to visualize the binding sites of the biotinylated lectins (Kirkeby et al., 1991). Localization of activity for myosin ATPase was performed according to Round et al. (1981) and activity for SDH was shown according to Kitkeby et al. (1988). Staining for Lectin Binding on Nitrocellulose Paper. After washing with TBS the blots were: (a) incubated for 24 hr at 4’C with biotinylated lectins diluted to a concentration of 10 pg/ml with TBS containing 1 mM CaCIz, MnC12, MgC12 and 0.05% Tween-20; (b) rinsed twice for 5 min in TBS buffer; (c) incubated for 30 min at room temperature with alkaline phosphatase-conjugatedavidin (Dakopatts 365; Glomp, Denmark) diluted 1:600with TBS; (d) rinsed three times for 5 min in TBS with 2.5 mg levamisole (Sigma L 9756; St Louis, MO) per 10 ml; (e) incubated for 15 min at room temperature in 10 ml Tris buffer (pH 9.5, 0.1 M), 10 mM MgCIz, 2.5 mg 5-bromo-4-chloroindoxyl phosphate (Bachem M-1780; Torrance, CA) in 0.1 ml N,N-dimethylformamide,3 mg nitroblue tetrazolium (Sigma N 6876), and 2.5 mg levamisole. For controls, 2 pl of the Xis buffer were applied to the nitrocellulose paper incubated with the different lectins and stained for possible lectin binding. In other experiments the myosin preparation and the muscle sections were incubated in buffer without lectin and stained for possible nonspecific avidin-biotin-alkaline phosphatase reaction.
Results The staining pattern after lectin incubation was very complex in muscle sections from the 60-day rats. Reaction could be recorded in different sites such as sarcoplasm, sarcolemma, connective tissue, and capillaries. A strong or moderate sarcoplasmic reaction, with the same staining in all fibers in the three muscles, was no-
Incubation with ConA, GS-11, HPA, and jacalin resulted in a mosaic staining pattern, with differences in the lectin staining intensity among individual fibers. The reaction was present as discrete granules and as diffuse cytoplasmic staining. Of the muscle investigated, the mosaic staining pattern was most apparent in the gracilis muscle. In this muscle a fiber was stained with the same intensity (strong or weak) after incubation with ConA, HPA, and GS-11, whereas incubation with jacalin resulted in a reversed reaction in most fibers. Thus, by comparing serial sections, a fiber that showed high affinity to ConA, HPA, and GS-I1 was weakly stained with jacalin, and fibers that were jacalin positive were weakly stained with the three other lectins (Figures la-Id). The muscle contained both Type I fibers (low activity for ATPase after incubation at pH 9.4) and Type I1 fibers (high activity for ATPase after incubation at p H 9.4) (Figure le). Incubation for SDH showed that both “white” fibers (low SDH activity) and “red” fibers (high SDH activity) were present (Figure lf). Serial sections that were incubated to show activity for lectins, ATPase, and SDH revealed that there was no relation between the lectin staining pattern and the intensity of enzyme activity in the fibers (Figures la-If). In the soleus muscle the staining reactions in the fibers after incubation with the different lectins was more homogeneous, with many fibers showing a moderate intensity. However, it was obvious also in this muscle that most of the fibers that were strongly ConA, GS-11, and HPA positive were jacalin negative, and vice versa (Figures 2a-2d). Again, there was no relation between the reactions for the lectins and the activity of myosin ATPase. In the superficial part of the masseter muscle, most fibers were stained moderately after incubation with ConA, GS-11, HPA, and jacalin. In the deep part of this muscle there appeared to be a more diverse reaction. In particular, incubation with GS-I1 resulted in a differentiated staining pattern, with some fibers reacting weakly and other fibers showing moderate or strong staining (Figure 2e). In contrast, the majority of the fibers were moderately stained with ConA (Figure 2f) and moderately to weakly with HPA and jacalin. All fibers were strongly ATPase positive. In the 10-day-oldrats, incubation with GS-I1resulted in strongly, moderately, and weakly stained muscle fibers (Figure 3a), while there was moderate staining in all the fibers of the three muscles examined after incubation with HPA, jacalin, and ConA (Figures 3b-3d). This means that in these young rats a lectin mosaic staining pattern could only be observed after incubation with GS-11. The lectin staining was mostly present as coarse granules, often as a part of a network as shown with ConA (Figure 3e). In the gracilis and soleus muscles a few fibers showed weak activity for myosin ATPase (Type I), whereas all the other fibers displayed a strong enzyme reaction (Type 11) (Figure 3f). In the control sections incubated without lectins there was very weak cytoplasmic staining. When dot-blots of the muscle extracts were incubated with lectins, ConA and HPA gave rise to a strong reaction, while weak staining appeared after incubation with GS-I1 and jacalin (Figure 4). In the blot incubated i n buffer without lectin and thereafter incubated with avidin-phosphatase, there was very weak staining for alkaline phosphatase (Figure 4).
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Electrophoretically separated muscle extracts were blotted to nitrocellulose paper and incubated with ConA, GS-11, HPA, and jacalin. Lectin-stained bands were obtained after incubation with ConA, GS-I1 and HPA, whereas no bands were present after incubation with jacalin (Figure 5 ) . The molecular weights of the ConAstained bands ranged from 120,000 to 43,000, while the bands stained with GS-I1 and HPA ranged from 100,000 to 43,000. Although many bands were HPA and ConA positive, only three GSI1 bands were clearly expressed. The apparent molecular weights of the GS-I1 bands were 100,000,90,000,and 43,000. These bands appeared also to be ConA and HPA positive. No nonspecific bands were observed when electrophoretic blots were incubated for reaction with avidin-alkaline phosphatase.
Discussion In the muscle sections a number of lectins detected sarcoplasmic
carbohydrate constituents. The exact localizations of the intracellular lectin binding sites are not known but in normal muscles most if not all of these glycoconjugates are membrane bound or are part of the myofibrillar proteins, since a membrane-deprived high-speed muscle supernatant contains virtually no soluble lectin binding glycoproteins (Kirkeby et al., 1992). Four of the lectins tested resulted in a sarcoplasmic mosaic staining pattern in the sections from adult rat muscle. These were ConA, HPA, GS-11, and jacalin. It is interesting that all four lectins have different sugar preferences. ConA, PSA, and LCA were the three mannoselglucose agglutinins tested in this study. Whereas incubation with ConA resulted in a mosaic staining in the sections of the gracilis, and partly in the soleus, LCA and PSA stained all fibers with the same intensity in these muscles. That PSA and LCA detect fibers that are not stained with ConA could be due to the broader pattern of sugar agglutination of the two former lectins. In contrast to ConA, LCA
Figure 1. (a-d) Lectin staining and (0-1) enzyme activity in gracilis muscle from 60-dayold rat. (a) ConA; (b) HPA; (c) GS-11; (d) jacalin. A mosaic staining pattern in the muscle section is evident. The same fibers that react strongly after incubation with ConA, HPA, and GS-II are weakly stained after incubation with jacalin and vice versa. (e) Myosin ATPase: (1) SDH. a-1 are serial sections. Note that there is no connection between lectin staining and enzyme reaction in the muscle fibers. Original magnification x 120. Bar = 50 pm.
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Figure 2. Lectin staining in (a-d) soleus muscle and (e,f) masseter muscle from 60day-old rat. (a) ConA; (b)GS-II; (c) HPA; (d) jacalin. In the soleus muscle the mosaic staining pattern for the lectins is less evident than in the gracilis muscle. (e) GS-II; (f) ConA. The mosaic staining for the lectins in the masseter muscle is evident only after incubationwith GS-II. Original magnification x 120. Bar = 50 wm.
and PSA might also detect N-acetylglucosamine (provided a fucose residue is present in the C-6 position) (Debray et al.. 1983). GS-I1 is an N-acetyl-fi-D-ghcosaminyl agglutinin. In a previous report we described a concordance between the staining intensity for GS-I1 and ConA and the PAS reaction in fibers from the rat gracilis muscle (Kirkeby et al., 1991). It was suggested that GSI1 detects muscle glycogen or glycoproteins attached to glycogen and ConA detects the same but also unidentified sarcoplasmic glycoproteins. Among the GalNAc-specificlectins (HPA, DBA, and SBA), only incubation with HPA gave rise to a sarcoplasmic mosaic staining pattern. Furthermore, HPA also detected the sarcolemma, whereas incubation with DBA and SBA resulted only in very weak, homogeneously disturbed sarcoplasmic reaction. The specificity of SBA is high for GalNAcal+3Gal (human blood group A). DBA detects A determinant and the Forssman-active pentasaccharide
(GalNAca- GalNAcP- 3Galal+4Galfil+4Glr,. while HPA agglutinates A, F, and Tn determinants [GalNAcal+O to Ser(Thr) of the protein core] (Wu et al., 1988). The Galfi+ specific lectin jacalin also gave rise to a mosaic staining pattern in the muscle sections, but the distribution tended to be the reverse of that obtained after HPA incubation. The other Gal-specific lectins, PNA and GS-I, did not detect sarcoplasmic constituents. Both PNA and jacalin have high specificity and af,finity for GalPl+3GalNAc, which forms the core disaccharide of 0-linked oligosaccharides (T antigen) (Wu et al.. 1988). The mosaic lectin staining pattern was not equally expressed in the three muscles investigated. It was evident in the gracilis, less distinct in the soleus, and in the masseter it was evident only for GS-11. These differences in the lectin staining pattern might be correlated with differences in muscle function. Gracilis is fast twitch, soleus is slow twitch, and masseter is a masticatory muscle which +
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Figure 3. ( a d ) Lectin staining and (f) ATPase activity in gracilis muscle from 10-dayold rats. (a) GS-II; (b) HPA; (c) jacalin; (d) ConA. There is variation in the staining intensity after incubation with GS-11, while all fibers react moderatelyafter incubationwith ConA. HPA, and jacalin. (e) A higher magnification of ConA staining in the muscle fibers. Note the network-likestaining pattern. (1) ATPase reaction in the muscle fibers. Most fibers are strongly stained, but a few fibers are ATPase negative. Original m a g nifications: a-d,f x 204; e x 985. Ears: ad,f = 20 pm; e = 5 pm.
both functionally and enzymatically (myosin ATPase) is different from limb muscles. In the 10-day-old rats no mosaic lectin staining pattern was observed in any of the muscles investigated after incubation with ConA, HPA, or jacalin. At this age the differentiation of the muscle fibers into the mature, specific types had barely started, since almost all the fibers were equally strongly stained after incubation for myo-
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Figure 4. Dot-blotof the muscle extract from hindleg muscles stained for lectin binding. (A) ConA; (e)GS-II; (C) HPA; (0)jacalin; (E) blank (incubated in buffer without lectin).
sin ATPase. At this stage of muscle specialization the muscles can all be described as slow. Furthermore, after lectin incubation the colored sarcoplasmic reaction product in the muscle fibers from 60-day-old rats was present as discrete granules and as a diffuse cytoplasmic reaction. In the very young rats the sarcoplasmic reaction consisted mainly of coarse granules, often as part of a network. Therefore, the lectin-staining patterns in very young and in more mature rat muscles are different, with a much more complex pattem in older muscles indicating a connection between muscle maturation and the presence of sarcoplasmic lectin binding sites. Using a fucose-specific lectin, Faze1 et al. (1989) have shown that in the embryonic heart the expression of the fucosylated glycoconjugates is stage-dependent and is therefore probably genetically regulated. There was no concordance between fiber reactions for enzyme activity and lectin staining in the adult muscles. This is in contrast to Ymagami et al. (1985), who described a correlation between staining for myosin ATPase and ConA. However, their material was
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In conclusion, the lectin-staining pattern in skeletal muscle is very complex. It might be related to development, specialization, and function of the individual muscles. There is no relationship between the mosaic lectin staining pattern and that obtained after incubation for myosin ATF’ase or succinic dehydrogenase.
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Literature Cited
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Capaldi J. Dunn MJ, Scwry CA. Dubowitz V (1985): Lectin binding in human skeletal muscle: a comparison of 15 different kctins. Histochcm J 1791 Debray H. Pierce-Creta1 A. Spik G, Montrevil J (1983): Affinity of ten insolubilized lectins towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides. In Beg-Hanscn TC, Spcnglcr G, eds. Lectins. biology, biochemistry. clinical biochemistry. Vol3. Berlin. de Gruytcr, 335 Faze1 AR. Sumida H. Schulte BA. Thompson RP (1989): Lectin histochcmistry of the embryonic heart: fucose-specific lectin binding sites in dcveloping rats and chicks. Am J Anat 184:76
Figure 5. Lectin staining of electrophoreticallyseparated and blotted muscle proteins. (A) ConA; (E) GS-II; (C) HPA (D) jacalin; (E) Fast Green; (F)molecular weight of marker proteins (in kilodaltons) stained by Fast Green.
Kirkcby S, Bag-Hanscn TC. Moe D. Garbarsch C (1991): Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures. Histochem J 23:345
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post-fixed in Carnoy’ssolution and pre-treated with trypsin before the incubation with lectins. It is likely that with this treatment other carbohydrate structures are exposed for reaction with ConA than those accessible in fresh untreated muscle, as in our material. ConA, HPA. and GS-I1stained sarcoplasmic components in the same fibers in leg muscles. In a blot of electrophoretically separated muscle proteins, three protein bands appeared to be detected by ConA, HPA, and GS-11. It might be suggested that these electrophoretically demonstrable lectin-positive proteins are at least partly responsible for the histochemically demonstrable lectin reaction. In addition, a number of protein bands were stained with ConA and HPA. and a band with apparent molecular weight of 120,000 seems to be specific for ConA binding. No bands were showed to have jacalin affinity, either because jacalin-positive glycoproteins are not present in the blot after electrophoresis or because the glycoconjugates positive for jacalin are glycolipids unable to migrate in the polyacrylamide gel.
Kirkcby S. Garbanch C, Matthiessen ME, Beg-Hansen TC.Moe D (1992): Changes of soluble glycoproteins in dystrophic (dy/dy)mouse muscle shown by lectin binding. Pathobiology. in press Kirkeby S. Moe D. Vilmann H (1988): Esterase profile of human masseter muscle. J Anat 157:79 Moggio M. Jann S. Adobbati L, Prcllc A. Gallanti A. Fagiolari G, Pellegrini G, Scarlato G (1989): Ultrastructural localization of calcium binding sites on human muscle cell surface. Muscle Nerve 12:910 Round JM, Matthcws Y, Jones DA (1981): A quick, simple and reliable histochemical method for ATPase in human muscle preparations. Histochcm J 12:707 Voit T, Patcl K. Sewry CA, Strong PN. Dubowitz V, Dunn MJ (1989): Membran-verandrungen bei DuchennelBecker-Muskeldystrophie:Lectinbindung und Dystrophin-lokalisation.Monatsschr Kinderheilkd 137:20 Wu AM, Sugii S. Herp A (1988): A guide for carbohydrate specificities of lectins. In Wu AM, cd. The molecular immunology of complex carbohydrates. Ncw York, London, Plenum Press. 819
Yamagami T, Hosaka M. Mori M (1985): Classification of skeletal muscle fibres by comparison of enzyme histochemistry with lectin binding. Cell Mol Biol 31:241
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