EXPERIMENTAL
NEUROLOGY
Glycolipid
(1975)
and Glycoprotein Sialyltransferase Enzyme Activity in Denervated Skeletal Muscle
JACK De~ayfmo~f
47, 381-391
MCLAUGHLIN
of Phaymacolog~ Mcdic.i,le and
AND
1%
BRUCE
md To.ricolog~. Unhwsity Dcrrfisfy.v. Rochrstcy, Ncso Rccrivrd
D~CCIII~WY
BOSMANN of Rochester
York
1 School
of
14642
19. 19iJ
Glycolipid and glycoprotein sialyltransferase enzyme activities were determined in homogenates of control and denervated rat extensor digitorum longus muscles. One week after denervation cytidine monophosphate-Nacetylneuraminic acid (CMP-NANA) : lactosyl ceramide and CMPNANA : fetuin sialyltransferase enzyme activities in denervated muscles were consistently higher in denervated than in control muscles, whether the activities were expressed on a protein, wet weight, or whole muscle basis. These findings support the hypothesis that control of muscle glycoconjugate metabolism, established in the normal relationship between nerve and muscle, is disrupted by denervatiun.
INTRODUCTION Mammalian cell membranes, including those of muscle, are enriched in carbohydrate-containing macromolecules (glycoconjugates) (8, 13, 25). Abood et al. (1) reported that each of the three general classes of glycoconjugates (glycoproteins, glycolipids, and glycosaminoglycans) was present in a muscle surface membrane preparation. Zacks and his coworkers (24, 26) have confirmed and extended these findings using various biochemical and cytohistochemical techniques, and other groups have reported that glycoproteins and glycolipids are components of the sarcoplasmic reticulum (15. 20) as well. While the functional importance of these membrane consituents in muscle is not presently known, they may, by analogy with other tissues, be involved in a number of cell-cell and cell-agent recognition phenomena and 1 Dr. McLaughlin is a postdoctoral fellow of the Muscular Dystrophy Association of America. Dr. Bosmann is a Scholar of the Leukemia Society of America. This study was supported in part by Grants GM-l.5190 and CA-13220 from N.I.H. We thank Mr. Roger Gutheil and Mr. Kenneth R. Case for technical assistance. 381 Copyright All rights
0
1975 by Academic Press, Inc. of reproduction in any form reserved.
382
MCLAUGHLIN
AND
BOSMANN
may be important determinants of cellular responses to a wide variety of stimuli (8). Moreover, it is of interest that the profound alterations in physiological and chemosensitive muscle membrane properties accompanying denervation (11) are paralleled by changes in muscle ganglioside (18) and sialoprotein (2, 9) composition. We previously reported that denervation of rat extensor digitorum longus muscle (EDL) was soon followed by an abrupt increase in the activity of a number of glycosidases (19). The time course of the elevation of at least two enzyme activities, P-o-mannosidase and a-L-fucosidase, was dependent on the length of the distal nerve stump, indicating that the nerve influenced the postdenervation elevation of enzyme activities. We now report that both glycolipid and glycoprotein sialyltransferase enzyme activities are also increased in homogenates of denervated EDL. Taken together, these findings of increased activity of both glycoconjugate catabolic and synthetic enzymes suggest that alterations in the biosynthesis and degradation of muscle glycoconjugates may be among the earliest biochemical consequences of denervation. MATERIALS
AND
METHODS
Tissue Preparation. Adult (225-325 g) male rats of the Long-Evans hooded strain were anesthetized with sodium secobarbital (40 mg/kg, ip). The EDL of the left or right hind leg was denervated by transecting the nerve just external to the peroneal muscle group. A sham operation was performed on the opposite leg and its EDL served as a control. No antibiotics were administered; the incisions were sutured, and the animals were maintained for one week on a regular laboratory diet with ad lib. access to water, as previously described (19). Animals were killed by a sharp blow on the neck and the EDL of each leg was removed and placed in a beaker of ice-cold saline solution. Each muscle was blotted twice on filter paper and weighed. Muscles were then minced in 12 vol of distilled water and thoroughly homogenized for 30 strokes with a Ten Broeck homogenizer held in an ice water bath. This homogenate was used for the assay of enzyme activities. Protein. Protein was determined by a slight modification of the method of Lowry et al. (16) using bovine serum albumin as a standard. Glycolipid: N-Acetylneurawzinic Acid Sialyl Transferase. Cytidine monoacid (CMP-NANA) : lactosyl ceramide phosphate-N-acetylneuraminic sialyltransferase enzyme activity was assayed by a modification of the method of Keenan, MorrC, and Basu (12). The complete reaction mixture in a final volume of 0.20 ml contained : N-stearoyl-dihydrolactocerebroside (Miles Laboratories), 0.25 pmole ; CMP-NANA (New England Nuclear,
DENERVATED
MUSCLE
383
CMP- [4, 5, 6, 7, 8, 9-l*C] NANA, 217 pCi/pmole), 0.7 nmole ; Tween 80 : Triton CF-54 ( 1 : 2, w/w), 1.2 mg ; sodium cacodylate buffer (pH 6.3), 30 pmole; Mn&, 2 rmole ; and 0.4 to 0.8 mg homogenate protein. After incubation for 1 hr at 30 C in a Dubnoff metabolic shaker, the reaction was terminated by the addition of 0.20 ml of methanol. Aliquots of the resulting mixtures were applied to Whatman 3’MM paper and dried. Paper chromatograms were developed (descending) for 16 hr in 1% sodium tetraborate at room temperature. The chromatograms were then dried and the origin cut out and incubated at 37 C for 1 hr with 15 ml of chloroform : methanol : water (2 : 1 : 0.2, v/v). The extracts were evaporated and radioactive products were quantitated by liquid scintillation spectrometry. Glycoprotein: N-Acetylnewah~ic Acid Transferase. The glycoprotein exogenous acceptor used in these experiments was fetuin minus sialic acid prepared by removal of sialic acid from native fetuin by acid hydrolysis as described (5). The complete system for the assay of fetuin : sialyltransferase activity contained in 0.20 ml final volume : desialyzed fetuin, 1.5 mg ; sodium cacodylate buffer (pH 5.4), 30 pmole: Triton X-100, 0.057,; CMP-NANA, 0.7 nmole; and 0.2 to 0.4 mg homogenate protein. Incubations were performed at 30 C for 0.5 hr and terminated by addition of 1% phosphotungstic acid in 0.5 N HCI. After centrifugation for 5 min at 2OOOg,pellets were washed twice with 10% trichloroacetic acid and once with ethanol : ether (2: 1, v/v). The washed pellets were then dissolved in 1 N NaOH and plated onto glass fiber discs. Radioactive products were quantitated by liquid scintillation spectrometry. Properties of Enzymatic Reaction Assays. Product formation in both glycolipid :- and glycoprotein : sialyltransferase assays was linear in terms of time, homogenate protein, and essentially linear with the concentration of CMP-NANA. Lactosyl ceramide and desialyzed fetuin were present in saturating amounts. Product formation was eliminated by boiling homogenates prior to assay and was negligible in immediately terminated reaction mixtures or in reaction mixtures incubated in an ice water bath. Endogenous sialyltransferase activity (activity in the absence of added acceptor) was too low to be determined reliably with either assay. Assays were performed in triplicate and activities were corrected for the small incorporation into endogenous acceptors. Statistics. There was considerable variation among animals in the values of enzyme activities, but no differences were found in individual control animals when the left and right muscles were compared. For statistical comparison of experimental and control muscles two-tailed levels of significance were determined by using the Wilcoxon matched-pairs signedranks test (22).
384
MCLAUGHLIN
AND
BOSMANN
RESULTS The data in Table 1 indicate that EDL denervated for 7 days had lost on average 18% of its wet weight, and its protein concentration (mg protein/g wet weight) was 13% below control values. Changes of this magnitude complicate selection of an appropriate base on which to refer enzyme activity data, making comparisons of normal and denervated muscles difficult. However, as is shown below, sialyltransferase enzyme activities in denervated muscles were consistently elevated over control values whether the activities were expressed on a protein, wet weight, or whole muscle basis. When animals were matched for size, the range of muscle weight values from normal animals was similar to the range found for the control muscles in this study and there was no gross trend toward hypertrophy of the control muscle. As shown in Tables 2 and 3, CMP-NANA: lactosyl ceramide and CMP-NANA : fetuin sialyltransferase enzyme activities were consistently elevated in homogenates of denervated EDL. When the values were calculated on a protein basis CMP-NANA : lactosyl ceramide sialyltransferase activity was increased 143% over control values; on a wet weight basis, 130% ; and on a whole muscle basis, 95%. Corresponding values for the increase in CMP-NANA : fetuin sialyltransferase activity were 68% (protein), 51% (wet weight), and 25% (whole muscle). The data in Table 4 show that CMP-NANA: lactosyl ceramide sialyltransferase activity was essentially additive when homogenates of deTABLE WET
Animal
8 f
AND PROTEIN CONCENTRATION FOR SEVEN DAYS~ Wet Denervated
9 Mean
WEIGHT
SD
1
0.167 0.173 0.125 0.125 0.132 0.136 0.143 0.160 0.154 0.146
f
0.018
D Protein concentration is milligrams are significant at the 0.01 level.
weight
OF EDL
(g) Control
0.210 0.199 0.124 0.166 0.169 0.159 0.191 0.193 0.191 0.178
f
of protein/g
DENERVATED
Protein concentration Denervated Control 12.5 12.5 95 107 108 111 109 112 105 111 f
0.026 wet
weight
of muscle.
9
131 129 128 123 12.5 126 125 130 122 127 f Differences
3
a Values
Mean
Ll L2 L3 L4 L5 L6 L7 L8
f
Animal
given
SD
are pmoles
0.08
of CMP-NANA
0.32 0.19 0.31 0.32 0.47 0.40 0.35 0.39 0.34 f
Per milligram Denervated
CMP-NANA:LACTOSYL
incorporated
zk 12.03
f
are all significant
13.56 12.34 15.66 20.30 24.41 19.51 19.66 15.09 17.57 4.06
IN DENERV~TED
wet weight Control
ACTIVITV
Differences
39.52 23.91 28.86 33.99 50.52 43.97 61.62 40.46 40.36 per hour.
2
Per gram Denervated
SIALYLTRANSFERASE
0.10 0.10 0.12 0.17 0.20 0.16 0.13 0.12 0.14 zk 0.04
protein Control
CERAMIDE
TABLE
at the 0.01
level.
4.13 3.10 4.03 2.88 3.09 f
6.68 5.98 10.59 6.23 6.01
Control 2.84 2.4.5 1.94 3.36
2.21
Per muscle
EDLa
6.59 4.14 3.62 4.26
f
Denervated
AND CONTROL
0.74
5 x Li
3 P y” g
l%
a Values
SD
given
Ll L2 L3 L4 L5 L6 L7 L8 Mean k
Animal
are pmoles
0.84
of CMP-NANA
6.01 6.60 8.02 8.01 7.14 6.68 8.46 7.08 7.25 f
Per milligram Denervated
CMP-NANA:FETuIN
incorporated
3.82 4.19 3.80 4.43 4.03 4.78 4.77 4.72 4.32 zk 0.42
protein Control
per hour.
SIALYLTRANSFERASE
3
Differences
500 540 487 545 503 602 613 620 5.51 f are all significant
750 826 759 8.59 778 738 947 1000 832 zt 97
wet weight Control
IN DENERVATED
Per gram Denervated
ACTIVITY
TABLE
54
125 143 9.5 108 102 101 151 140 121 f
Denervated
EDLa
at the 0.01 level.
AND CONTROL
22
Per muscle
105 107 60 90 85 96 118 114 97 f
Control
19
DENERVATED TABLE CMP-NANA:LACTOSYL MIXTURES
Experiment
4
CERAMIDE OF HOMOGENATES CONTROL
SIALYLTRANSFERASE FROM DENERVATED EDLa
Homogenate b-+x)
1
2
3
4
a Amounts respectively.
387
MUSCLE
of homogenates
0.474 0.634 0.237
D alone C alone D + 0.317
0.538 0.750 0.269
D alone C alone D + 0.375
0.602 0.800 0.301
D alone C alone D + 0.400
0.574 0.790 0.287
D alone C alone D + 0.395
are as protein.
ASSAYS ON AND
pmol/hr
C
C
C
C
0.145 0.078 0.111 0.112
(found) (theoretical)
0.171 0.134 0.155 0.153
(found) (theoretical)
0.267 0.165 0.238 0.216
(found) (theoretical)
0.239 0.124 0.183 0.182
(found) (theoretical)
D and C are denervated
and control
EDL,
nervated and control muscles were mixed. Similar results were found for CMP-NANA : fetuin sialyltransferase activity. These experiments do not support the notion that a change in the concentration of free activators, inhibitors, cofactors, etc., accounted for the elevated enzymatic activities. This point is discussed in more detail below. Lastly, we have now begun experiments to characterize in detail the nature of the elevations in glycolipid : and glycoprotein : sialyltransferase activities. Preliminary results indicate that denervation did not lead to changes in enzyme activity dependence on pH or temperature for either CMP-NANA : lactosyl ceramide or CMP-NANA : fetuin sialyltransferase. The apparent Km values for CMP-NANA, lactosyl ceramide, and desialyzed fetuin at optimal assay conditions were not altered by denervation. Reaction products have been tentatively identified and seem qualitatively unchanged with denervation.
388
MCLAUGHLIN
AND
BOSMANN
DISCUSSION To our knowledge this is the first report of glycolipid :- or glycoprotein :sialyltransferase enzyme activity in skeletal muscle. The study was first prompted by a report (18) that denervation of soleus or gastrocnemius muscles of rabbits, cats, or rats brought about an absolute increase in the ganglioside content of the muscles. That increase in rabbits, determined 8 days after denervation, was largely the result of a 67% elevation in the amount of hematoside (GM3), the major ganglioside of skeletal muscle (14, 18, 20, 23). Furthermore, in viva incorporation studies with N-acetyl[3H]n-mannosamine, a precursor of the sialic acid moiety of gangliosides, demonstrated a 47% increase in the labeling of GM3 in denervated muscle. No significant differences were found in other gangliosides studied, and the authors concluded that the increased content of GM3 in denervated muscle was the result of de nova synthesis. In a subsequent report Max (17) found no increase in the GM3 content of rat gastrocnemius muscle rendered atrophic by skeletal fixation. The muscle CMP-NANA : glycolipid sialyltransferase enzyme activity studied in the present report and found elevated in homogenates of denervated EDL catalyzed the in vitro biosynthesis of GM3 (N-acetylneuraminylgalactosylglucosylceramide) from lactosylceramide and CMPNANA. Similar enzymatic activity has been detected in brain (3, 4, 7) and in that tissue the more complex gangliosides are likely synthesized from GM3 by the sequential addition of monosaccharide and sialic acid moieties from sugar nucleotide donors (21). Gangliosides more complex than GM3 are also present in skeletal muscle (14, 18, 20, 23) and while quantitative and qualitative differences in composition exist, the major steps in the formation of muscle gangliosides may be similar to those in brain. The increased amounts of GM3 in denervated muscle reported by Max, Nelson and Brady (18) could result from: (i) increased biosynthesis of GM3, (ii) decreased catabolism of GM3, or (iii) decreased biosynthesis of more complex gangliosides for which GM3 is a precursor. The possibility that biosynthesis of GM3 is increased as a result of denervation receives strong support from our finding that CMP-NANA:lactosyl ceramide sialyltransferase activity is elevated in homogenates of denervated muscle. It should be pointed out, however, that an in vitro assay system was used in these studies and the results must be cautiously extrapolated to the situation in V&O. Assay conditions were optimized for the enzymatic formation of GM3 and while homogenates of denervated EDL consistently exhibited an increased synthetic capacity, synthesis in the cell may be influenced by the availability of precursors and effecters or by the structural relationships of enzyme and substrates, etc.
DENERVATED
Id USCLE
389
As discussed previously, Max, Nelson and Brady (18) reported that when N-acetyl- [ 3H]D-mannosamine was incorporated into denervated muscle gangliosides, the specific activities of gangliosides other than GM3 were not greatly different from control values (range 106 to 111% of control values). This could be taken as tentative evidence against the third possibility but the point should be further examined. We know of no study that deals with the catabolism of GM3 in skeletal muscle and the second possibility cannot be excluded at present. Duffard and Caputto (IO) found an inhibitor of CMP-NANA :lactosyl ceramide sialyltransferase in rat brain and observed that the inhibitor increases with the age of the animal. The mixing experiments shown in Table 4 are not consistent with the presence of such an inhibitor in EDL homogenates, but the point should be investigated more thoroughly by kinetic methods. Moreover, stable complexes formed between enzymes and effecters would not be detected in the mixing experiments. The sialoglycoprotein concentration of rabbit gastrocnemius muscle and muscle membrane preparations from rat EDL. anterior tibialis, and gastrolcnemius muscles was also reported to be increased after denervation (2, 9)) but no data were given for particular sialoglycoprotein species. With similar reservations about the comparison of irz vitro and in vivo biochemical data discussed previously, these results and the present demonstration of elevated CMP-NANA : fetuin sialyltransferase activity suggest that the capacity for sialoglycoprotein biosynthesis is also enhanced in denervated muscle. The preparation of various endogenous acceptors from whole muscle or muscle membrane preparations for the purpose of assaying sialyland other muscle glycosyltransferase activities may help clarify this point. We are not aware of any report describing muscle neuraminidase activity active with sialylglycoprotein as substrate. The possibility of decreased catabolism of muscle sialoglycoproteins in denervated muscle cannot be presently excluded. An understanding of the molecular basis of the membrane alterations in denervated skeletal muscle is, of course, hampered by the paucity of information available concerning structure-function relationships in the sarcolemma and sarcoplasmic reticulum. One approach to these problems can be made by investigating the metabolism of macromolecular constituents of muscle membrane systems in normal and denervated skeletal muscle. A study by Bunch et al. (6) on the incorporation of 3ZPi and [2-SH]glycerol into membrane glycerophosphatides of denervated muscle and the previously discussed work of Max ( 18), Appel (2), and coworkers are noteworthy in this regard. We previously reported (19) that denervation of rat EDL was rapidly followed by increased activity of glycosidase, acid phosphatase, and acid proteolytic enzymes, and on the basis of this and other evidence we suggested that the activity of enzymes likely to be
390
MCLAUGHLIN
AND
BOSMANN
involved in the catabolism of membrane macromolecules was to some extent controlled by neural influences. The demonstration in the present report that the activities of glycolipid and glycoprotein : sialyltransferases are also altered by denervation suggests that control of membrane glycoconjugate biosynthesis may be disrupted as well. The detailed investigation of these phenomena, focusing on particular membrane components (e.g., GM3), may provide insight into the nature of the changes brought about in the membrane properties of denervated muscle and into the larger related problem of nerve-muscle (“trophic”) relationships ( 11). REFERENCES 1.
2.
3.
4. 5.
6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16.
L. G., K. KURAHASI, E. BRUNNGRABER, and K. KOKETSU. 1966. Biochemical analysis of isolated bullfrog sarcolemma. Biochim. Biophys. Acta 112 : 330-339. ANDREW, C. G., and S. H. APPEL. 1973. Macromolecular characterizatioa of muscle membranes. I. Proteins and sialic acid of normal and denervated muscle. J. Biol. Chem. 248 : 5156-5163. ARCE, A., H. F. MACCIONI, and R. CAPUTTO. 1966. Enzymic binding of sialyl groups to ganglioside derivatives by preparations from the brain of a young rat. Arch. B&hem. Biophys. 116: 52-58. BAN, S. 1966. “Studies on the Biosynthesis of Gangliosides.” Ph.D. thesis, University of Michigan, Ann Arbor, Mich. BOSMANN, H. B. 1973. Synthesis of glycoproteins in brain: identification, purification and properties of a synaptosomal sialyl transferase utilizing endogenous and exogenous acceptors. J. Nezrrochem. 20: 1037-1049. BUNCH, W., G. KALLSEN, J. BERRY, and C. EDWARDS. 1970. The effect of denervation on incorporation of 32P and [?H]glycerol by the muscle membrane. J. Neurochem. 17: 613620. CAPUTTO, R., H. J. MACCIONI, and A. ARCE. 1974. Biosynthesis of brain gangliosides. Mol. Cell. Biochem. 4: 97-106. COOK, G. M. W., and R. W. STODDART. 1973. “Surface Carbohydrates of the Cell.” Academic Press, London. COTRUFO, R., and S. H. APPEL. 1973. Effects of denervation on glycoproteins of rabbit gastrocnemius and soleus muscles. Exfi. Neurol. 39: S-69. DUFFARD, R. O., and R. CAPUTTO. 1972. A natural inhibitor of sialyl transferase and its possible influence on this enzyme activity during brain development. Biochemistry 11: 1396-1400. GUTH, L. 1968. “Trophic” influences of nerve on muscle. Phgsiol. Rev. 48 : 64.5687. KEENAN, T. W., D. J. MORRI?, and S. BAN. 1974. Ganglioside biosynthesis. Concentration of glycosphingolipid glycosyltransferases in Golgi apparatus from rat liver. J. Biol. Chem. 249: 310-315. KRAEMER, P. M. 1971. Complex carbohydrates of animal cells: biochemistry and physiology of the cell periphery. Biomembranes 1: 67-190. LASSAGA, F. E., I. ALBARRACIN, and R. CAPUTTO. 1967. Gangli6sidos de1 tejido muscular. Atrnn Assoc. Q&Z. Argent. 55: 309-315. LOUIS, C., and E. M. SHOOTER. 1972. The proteins of rabbit skeletal muscle sarcoplasmic reticulum. Arch. Biochem. Biophys. 153: 641-655. LOWRY, 0. H., H. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. ABOOD,
DENERVATED
MUSCLE
391
17. MAX, S. R. 1971. Neural regulation of muscle ganglioside biosynthesis. Society for Neuroscience First Annual Meeting, Abstracts, p. 145. Oct. 27-30, Washington, D. C. 18. MAX, S. R., P. G. NELSON, and R. 0. BRADY. 1970. The effect of denervation on the composition of muscle gangliosides. J. Newochcm. 17: 1517-1520. 19. MCLAUGHLIN, J., L. G. ABOOD, and H. B. BOSMANN. 1974. Early elevations of glycosidase, acid phosphatase, and acid proteolytic enzyme activity in denervated skeletal muscle. Exp. Nmrol. 42: 541-554. 20. NARASIMHAM, R., R. K. MURRAY, and D. H. MACLENNAN. lY74. Presence of glycosphingolipids in the sarcosplasmic reticulum fraction of rabbit skeletal muscle. FEBS Lett. 43 : 23-26. 21. ROSEMAN, S. 1970. The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. Cherrz. Phys. Lipids 5 : 270-297. 22. SIEGEL, S. 1956. “Nonparametric Statistics for the Behavioral Sciences.” McGrawHill, New York. 23. SVENNERHOLM, L., A. BRUCE, J.-E. MANSSON, B.-M. RYNMARK, and M.-T. VANIER. 1972. Sphingolipids of human skeletal muscle. Bioch% Bioplzys. =Icta 280 : 62&-636. 24. VANDENBURGH, H. H., M. F. SHEFF, and S. I. ZACKS. 1974. Chemical composition of isolated rat skeletal sarcolemma. J. Membrane Biol. 17: l-12. 25. WINZLER, R. J. 1972. Glycoproteins of plasma membranes, pp. 1268-1287. Zpt “Glycoproteins. Their Composition, Structure, and Function, Part B.” A. Gottschalk [Ed.]. Elsevier, Amsterdam. 26. ZACKS, S. I., H. VANDENBURGH, and M. F. SIIEFF. 1973. Cytochemical and physical properties of myofiber external lamina. J. NiStOChEIII. Cytochrwz. 21 : 895-901.
NEUROLOGY
Glycolipid
(1975)
and Glycoprotein Sialyltransferase Enzyme Activity in Denervated Skeletal Muscle
JACK De~ayfmo~f
47, 381-391
MCLAUGHLIN
of Phaymacolog~ Mcdic.i,le and
AND
1%
BRUCE
md To.ricolog~. Unhwsity Dcrrfisfy.v. Rochrstcy, Ncso Rccrivrd
D~CCIII~WY
BOSMANN of Rochester
York
1 School
of
14642
19. 19iJ
Glycolipid and glycoprotein sialyltransferase enzyme activities were determined in homogenates of control and denervated rat extensor digitorum longus muscles. One week after denervation cytidine monophosphate-Nacetylneuraminic acid (CMP-NANA) : lactosyl ceramide and CMPNANA : fetuin sialyltransferase enzyme activities in denervated muscles were consistently higher in denervated than in control muscles, whether the activities were expressed on a protein, wet weight, or whole muscle basis. These findings support the hypothesis that control of muscle glycoconjugate metabolism, established in the normal relationship between nerve and muscle, is disrupted by denervatiun.
INTRODUCTION Mammalian cell membranes, including those of muscle, are enriched in carbohydrate-containing macromolecules (glycoconjugates) (8, 13, 25). Abood et al. (1) reported that each of the three general classes of glycoconjugates (glycoproteins, glycolipids, and glycosaminoglycans) was present in a muscle surface membrane preparation. Zacks and his coworkers (24, 26) have confirmed and extended these findings using various biochemical and cytohistochemical techniques, and other groups have reported that glycoproteins and glycolipids are components of the sarcoplasmic reticulum (15. 20) as well. While the functional importance of these membrane consituents in muscle is not presently known, they may, by analogy with other tissues, be involved in a number of cell-cell and cell-agent recognition phenomena and 1 Dr. McLaughlin is a postdoctoral fellow of the Muscular Dystrophy Association of America. Dr. Bosmann is a Scholar of the Leukemia Society of America. This study was supported in part by Grants GM-l.5190 and CA-13220 from N.I.H. We thank Mr. Roger Gutheil and Mr. Kenneth R. Case for technical assistance. 381 Copyright All rights
0
1975 by Academic Press, Inc. of reproduction in any form reserved.
382
MCLAUGHLIN
AND
BOSMANN
may be important determinants of cellular responses to a wide variety of stimuli (8). Moreover, it is of interest that the profound alterations in physiological and chemosensitive muscle membrane properties accompanying denervation (11) are paralleled by changes in muscle ganglioside (18) and sialoprotein (2, 9) composition. We previously reported that denervation of rat extensor digitorum longus muscle (EDL) was soon followed by an abrupt increase in the activity of a number of glycosidases (19). The time course of the elevation of at least two enzyme activities, P-o-mannosidase and a-L-fucosidase, was dependent on the length of the distal nerve stump, indicating that the nerve influenced the postdenervation elevation of enzyme activities. We now report that both glycolipid and glycoprotein sialyltransferase enzyme activities are also increased in homogenates of denervated EDL. Taken together, these findings of increased activity of both glycoconjugate catabolic and synthetic enzymes suggest that alterations in the biosynthesis and degradation of muscle glycoconjugates may be among the earliest biochemical consequences of denervation. MATERIALS
AND
METHODS
Tissue Preparation. Adult (225-325 g) male rats of the Long-Evans hooded strain were anesthetized with sodium secobarbital (40 mg/kg, ip). The EDL of the left or right hind leg was denervated by transecting the nerve just external to the peroneal muscle group. A sham operation was performed on the opposite leg and its EDL served as a control. No antibiotics were administered; the incisions were sutured, and the animals were maintained for one week on a regular laboratory diet with ad lib. access to water, as previously described (19). Animals were killed by a sharp blow on the neck and the EDL of each leg was removed and placed in a beaker of ice-cold saline solution. Each muscle was blotted twice on filter paper and weighed. Muscles were then minced in 12 vol of distilled water and thoroughly homogenized for 30 strokes with a Ten Broeck homogenizer held in an ice water bath. This homogenate was used for the assay of enzyme activities. Protein. Protein was determined by a slight modification of the method of Lowry et al. (16) using bovine serum albumin as a standard. Glycolipid: N-Acetylneurawzinic Acid Sialyl Transferase. Cytidine monoacid (CMP-NANA) : lactosyl ceramide phosphate-N-acetylneuraminic sialyltransferase enzyme activity was assayed by a modification of the method of Keenan, MorrC, and Basu (12). The complete reaction mixture in a final volume of 0.20 ml contained : N-stearoyl-dihydrolactocerebroside (Miles Laboratories), 0.25 pmole ; CMP-NANA (New England Nuclear,
DENERVATED
MUSCLE
383
CMP- [4, 5, 6, 7, 8, 9-l*C] NANA, 217 pCi/pmole), 0.7 nmole ; Tween 80 : Triton CF-54 ( 1 : 2, w/w), 1.2 mg ; sodium cacodylate buffer (pH 6.3), 30 pmole; Mn&, 2 rmole ; and 0.4 to 0.8 mg homogenate protein. After incubation for 1 hr at 30 C in a Dubnoff metabolic shaker, the reaction was terminated by the addition of 0.20 ml of methanol. Aliquots of the resulting mixtures were applied to Whatman 3’MM paper and dried. Paper chromatograms were developed (descending) for 16 hr in 1% sodium tetraborate at room temperature. The chromatograms were then dried and the origin cut out and incubated at 37 C for 1 hr with 15 ml of chloroform : methanol : water (2 : 1 : 0.2, v/v). The extracts were evaporated and radioactive products were quantitated by liquid scintillation spectrometry. Glycoprotein: N-Acetylnewah~ic Acid Transferase. The glycoprotein exogenous acceptor used in these experiments was fetuin minus sialic acid prepared by removal of sialic acid from native fetuin by acid hydrolysis as described (5). The complete system for the assay of fetuin : sialyltransferase activity contained in 0.20 ml final volume : desialyzed fetuin, 1.5 mg ; sodium cacodylate buffer (pH 5.4), 30 pmole: Triton X-100, 0.057,; CMP-NANA, 0.7 nmole; and 0.2 to 0.4 mg homogenate protein. Incubations were performed at 30 C for 0.5 hr and terminated by addition of 1% phosphotungstic acid in 0.5 N HCI. After centrifugation for 5 min at 2OOOg,pellets were washed twice with 10% trichloroacetic acid and once with ethanol : ether (2: 1, v/v). The washed pellets were then dissolved in 1 N NaOH and plated onto glass fiber discs. Radioactive products were quantitated by liquid scintillation spectrometry. Properties of Enzymatic Reaction Assays. Product formation in both glycolipid :- and glycoprotein : sialyltransferase assays was linear in terms of time, homogenate protein, and essentially linear with the concentration of CMP-NANA. Lactosyl ceramide and desialyzed fetuin were present in saturating amounts. Product formation was eliminated by boiling homogenates prior to assay and was negligible in immediately terminated reaction mixtures or in reaction mixtures incubated in an ice water bath. Endogenous sialyltransferase activity (activity in the absence of added acceptor) was too low to be determined reliably with either assay. Assays were performed in triplicate and activities were corrected for the small incorporation into endogenous acceptors. Statistics. There was considerable variation among animals in the values of enzyme activities, but no differences were found in individual control animals when the left and right muscles were compared. For statistical comparison of experimental and control muscles two-tailed levels of significance were determined by using the Wilcoxon matched-pairs signedranks test (22).
384
MCLAUGHLIN
AND
BOSMANN
RESULTS The data in Table 1 indicate that EDL denervated for 7 days had lost on average 18% of its wet weight, and its protein concentration (mg protein/g wet weight) was 13% below control values. Changes of this magnitude complicate selection of an appropriate base on which to refer enzyme activity data, making comparisons of normal and denervated muscles difficult. However, as is shown below, sialyltransferase enzyme activities in denervated muscles were consistently elevated over control values whether the activities were expressed on a protein, wet weight, or whole muscle basis. When animals were matched for size, the range of muscle weight values from normal animals was similar to the range found for the control muscles in this study and there was no gross trend toward hypertrophy of the control muscle. As shown in Tables 2 and 3, CMP-NANA: lactosyl ceramide and CMP-NANA : fetuin sialyltransferase enzyme activities were consistently elevated in homogenates of denervated EDL. When the values were calculated on a protein basis CMP-NANA : lactosyl ceramide sialyltransferase activity was increased 143% over control values; on a wet weight basis, 130% ; and on a whole muscle basis, 95%. Corresponding values for the increase in CMP-NANA : fetuin sialyltransferase activity were 68% (protein), 51% (wet weight), and 25% (whole muscle). The data in Table 4 show that CMP-NANA: lactosyl ceramide sialyltransferase activity was essentially additive when homogenates of deTABLE WET
Animal
8 f
AND PROTEIN CONCENTRATION FOR SEVEN DAYS~ Wet Denervated
9 Mean
WEIGHT
SD
1
0.167 0.173 0.125 0.125 0.132 0.136 0.143 0.160 0.154 0.146
f
0.018
D Protein concentration is milligrams are significant at the 0.01 level.
weight
OF EDL
(g) Control
0.210 0.199 0.124 0.166 0.169 0.159 0.191 0.193 0.191 0.178
f
of protein/g
DENERVATED
Protein concentration Denervated Control 12.5 12.5 95 107 108 111 109 112 105 111 f
0.026 wet
weight
of muscle.
9
131 129 128 123 12.5 126 125 130 122 127 f Differences
3
a Values
Mean
Ll L2 L3 L4 L5 L6 L7 L8
f
Animal
given
SD
are pmoles
0.08
of CMP-NANA
0.32 0.19 0.31 0.32 0.47 0.40 0.35 0.39 0.34 f
Per milligram Denervated
CMP-NANA:LACTOSYL
incorporated
zk 12.03
f
are all significant
13.56 12.34 15.66 20.30 24.41 19.51 19.66 15.09 17.57 4.06
IN DENERV~TED
wet weight Control
ACTIVITV
Differences
39.52 23.91 28.86 33.99 50.52 43.97 61.62 40.46 40.36 per hour.
2
Per gram Denervated
SIALYLTRANSFERASE
0.10 0.10 0.12 0.17 0.20 0.16 0.13 0.12 0.14 zk 0.04
protein Control
CERAMIDE
TABLE
at the 0.01
level.
4.13 3.10 4.03 2.88 3.09 f
6.68 5.98 10.59 6.23 6.01
Control 2.84 2.4.5 1.94 3.36
2.21
Per muscle
EDLa
6.59 4.14 3.62 4.26
f
Denervated
AND CONTROL
0.74
5 x Li
3 P y” g
l%
a Values
SD
given
Ll L2 L3 L4 L5 L6 L7 L8 Mean k
Animal
are pmoles
0.84
of CMP-NANA
6.01 6.60 8.02 8.01 7.14 6.68 8.46 7.08 7.25 f
Per milligram Denervated
CMP-NANA:FETuIN
incorporated
3.82 4.19 3.80 4.43 4.03 4.78 4.77 4.72 4.32 zk 0.42
protein Control
per hour.
SIALYLTRANSFERASE
3
Differences
500 540 487 545 503 602 613 620 5.51 f are all significant
750 826 759 8.59 778 738 947 1000 832 zt 97
wet weight Control
IN DENERVATED
Per gram Denervated
ACTIVITY
TABLE
54
125 143 9.5 108 102 101 151 140 121 f
Denervated
EDLa
at the 0.01 level.
AND CONTROL
22
Per muscle
105 107 60 90 85 96 118 114 97 f
Control
19
DENERVATED TABLE CMP-NANA:LACTOSYL MIXTURES
Experiment
4
CERAMIDE OF HOMOGENATES CONTROL
SIALYLTRANSFERASE FROM DENERVATED EDLa
Homogenate b-+x)
1
2
3
4
a Amounts respectively.
387
MUSCLE
of homogenates
0.474 0.634 0.237
D alone C alone D + 0.317
0.538 0.750 0.269
D alone C alone D + 0.375
0.602 0.800 0.301
D alone C alone D + 0.400
0.574 0.790 0.287
D alone C alone D + 0.395
are as protein.
ASSAYS ON AND
pmol/hr
C
C
C
C
0.145 0.078 0.111 0.112
(found) (theoretical)
0.171 0.134 0.155 0.153
(found) (theoretical)
0.267 0.165 0.238 0.216
(found) (theoretical)
0.239 0.124 0.183 0.182
(found) (theoretical)
D and C are denervated
and control
EDL,
nervated and control muscles were mixed. Similar results were found for CMP-NANA : fetuin sialyltransferase activity. These experiments do not support the notion that a change in the concentration of free activators, inhibitors, cofactors, etc., accounted for the elevated enzymatic activities. This point is discussed in more detail below. Lastly, we have now begun experiments to characterize in detail the nature of the elevations in glycolipid : and glycoprotein : sialyltransferase activities. Preliminary results indicate that denervation did not lead to changes in enzyme activity dependence on pH or temperature for either CMP-NANA : lactosyl ceramide or CMP-NANA : fetuin sialyltransferase. The apparent Km values for CMP-NANA, lactosyl ceramide, and desialyzed fetuin at optimal assay conditions were not altered by denervation. Reaction products have been tentatively identified and seem qualitatively unchanged with denervation.
388
MCLAUGHLIN
AND
BOSMANN
DISCUSSION To our knowledge this is the first report of glycolipid :- or glycoprotein :sialyltransferase enzyme activity in skeletal muscle. The study was first prompted by a report (18) that denervation of soleus or gastrocnemius muscles of rabbits, cats, or rats brought about an absolute increase in the ganglioside content of the muscles. That increase in rabbits, determined 8 days after denervation, was largely the result of a 67% elevation in the amount of hematoside (GM3), the major ganglioside of skeletal muscle (14, 18, 20, 23). Furthermore, in viva incorporation studies with N-acetyl[3H]n-mannosamine, a precursor of the sialic acid moiety of gangliosides, demonstrated a 47% increase in the labeling of GM3 in denervated muscle. No significant differences were found in other gangliosides studied, and the authors concluded that the increased content of GM3 in denervated muscle was the result of de nova synthesis. In a subsequent report Max (17) found no increase in the GM3 content of rat gastrocnemius muscle rendered atrophic by skeletal fixation. The muscle CMP-NANA : glycolipid sialyltransferase enzyme activity studied in the present report and found elevated in homogenates of denervated EDL catalyzed the in vitro biosynthesis of GM3 (N-acetylneuraminylgalactosylglucosylceramide) from lactosylceramide and CMPNANA. Similar enzymatic activity has been detected in brain (3, 4, 7) and in that tissue the more complex gangliosides are likely synthesized from GM3 by the sequential addition of monosaccharide and sialic acid moieties from sugar nucleotide donors (21). Gangliosides more complex than GM3 are also present in skeletal muscle (14, 18, 20, 23) and while quantitative and qualitative differences in composition exist, the major steps in the formation of muscle gangliosides may be similar to those in brain. The increased amounts of GM3 in denervated muscle reported by Max, Nelson and Brady (18) could result from: (i) increased biosynthesis of GM3, (ii) decreased catabolism of GM3, or (iii) decreased biosynthesis of more complex gangliosides for which GM3 is a precursor. The possibility that biosynthesis of GM3 is increased as a result of denervation receives strong support from our finding that CMP-NANA:lactosyl ceramide sialyltransferase activity is elevated in homogenates of denervated muscle. It should be pointed out, however, that an in vitro assay system was used in these studies and the results must be cautiously extrapolated to the situation in V&O. Assay conditions were optimized for the enzymatic formation of GM3 and while homogenates of denervated EDL consistently exhibited an increased synthetic capacity, synthesis in the cell may be influenced by the availability of precursors and effecters or by the structural relationships of enzyme and substrates, etc.
DENERVATED
Id USCLE
389
As discussed previously, Max, Nelson and Brady (18) reported that when N-acetyl- [ 3H]D-mannosamine was incorporated into denervated muscle gangliosides, the specific activities of gangliosides other than GM3 were not greatly different from control values (range 106 to 111% of control values). This could be taken as tentative evidence against the third possibility but the point should be further examined. We know of no study that deals with the catabolism of GM3 in skeletal muscle and the second possibility cannot be excluded at present. Duffard and Caputto (IO) found an inhibitor of CMP-NANA :lactosyl ceramide sialyltransferase in rat brain and observed that the inhibitor increases with the age of the animal. The mixing experiments shown in Table 4 are not consistent with the presence of such an inhibitor in EDL homogenates, but the point should be investigated more thoroughly by kinetic methods. Moreover, stable complexes formed between enzymes and effecters would not be detected in the mixing experiments. The sialoglycoprotein concentration of rabbit gastrocnemius muscle and muscle membrane preparations from rat EDL. anterior tibialis, and gastrolcnemius muscles was also reported to be increased after denervation (2, 9)) but no data were given for particular sialoglycoprotein species. With similar reservations about the comparison of irz vitro and in vivo biochemical data discussed previously, these results and the present demonstration of elevated CMP-NANA : fetuin sialyltransferase activity suggest that the capacity for sialoglycoprotein biosynthesis is also enhanced in denervated muscle. The preparation of various endogenous acceptors from whole muscle or muscle membrane preparations for the purpose of assaying sialyland other muscle glycosyltransferase activities may help clarify this point. We are not aware of any report describing muscle neuraminidase activity active with sialylglycoprotein as substrate. The possibility of decreased catabolism of muscle sialoglycoproteins in denervated muscle cannot be presently excluded. An understanding of the molecular basis of the membrane alterations in denervated skeletal muscle is, of course, hampered by the paucity of information available concerning structure-function relationships in the sarcolemma and sarcoplasmic reticulum. One approach to these problems can be made by investigating the metabolism of macromolecular constituents of muscle membrane systems in normal and denervated skeletal muscle. A study by Bunch et al. (6) on the incorporation of 3ZPi and [2-SH]glycerol into membrane glycerophosphatides of denervated muscle and the previously discussed work of Max ( 18), Appel (2), and coworkers are noteworthy in this regard. We previously reported (19) that denervation of rat EDL was rapidly followed by increased activity of glycosidase, acid phosphatase, and acid proteolytic enzymes, and on the basis of this and other evidence we suggested that the activity of enzymes likely to be
390
MCLAUGHLIN
AND
BOSMANN
involved in the catabolism of membrane macromolecules was to some extent controlled by neural influences. The demonstration in the present report that the activities of glycolipid and glycoprotein : sialyltransferases are also altered by denervation suggests that control of membrane glycoconjugate biosynthesis may be disrupted as well. The detailed investigation of these phenomena, focusing on particular membrane components (e.g., GM3), may provide insight into the nature of the changes brought about in the membrane properties of denervated muscle and into the larger related problem of nerve-muscle (“trophic”) relationships ( 11). REFERENCES 1.
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