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Myelin-associated glycoprotein: A developmental change
506
Brain Research, 58 (1973) 506-509 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Myelin.associated glycoprotein: a developmental change RICHARD H. QUARLES, JOHN L. EVERLY AND ROSCOE O. BRADY Developmental and Metabolic Neurology Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)
(Accepted May 8th, 1973)
Glycoproteins on the plasma membranes of cells are believed to be involved in specific cell-cell interactions 8. Myelin which is thought to be derived from the plasma membrane of the oligodendrocyte has generally been considered to contain little or no glycoprotein. Recently, however, we have demonstrated a glycoprotein which is closely associated with myelin4,L In this paper, we present evidence for a developmental change in this glycoprotein which could play an important role in the process of myelination. A preliminary report of this finding has been presented 5. Sprague-Dawley rats of various ages were purchased from Taconic Farms (Germantown, New York). L-[1,5,6-aH]Fucose and L-[1-14C]fucose were obtained from New England Nuclear Corporation (Boston, Mass.). The animals were injected intracerebrally 3 with 20 #Ci of [3H]fucose (125 #Ci//~mole) or 8 #Ci of [14C]fucose (50/~Ci//zmole) and sacrificed 16 h later. Five percent homogenates of the brains from these animals were prepared in 0.32 M sucrose, and myelin was purified essentially according to the procedure of Norton and Poduslo ~. In most experiments, the myelin was purified from a mixed homogenate obtained by combining equal volumes of a [3H]fucose-labeled homogenate from animals of one age and a [14C]fucose-labeled homogenate from animals of another age. The myelin was lyophilized and dissolved in 2.5 ~ sodium dodecyl sulfate (SDS), 1 ~ sodium carbonate, and 10~ mercaptoethanol. It was dialyzed aganst 0 . 1 ~ SDS, 0.01 M sodium phosphate buffer (pH 7.2), 1.6 M urea, and 0.05 ~ dithiothreitol. The samples were electrophoresed on 5 ~ SDS-polyacrylamide gels (11 cm × 6 mm) prepared according to Shapiro et aLL After electrophoresis the gels were fractionated on a Mickle Gel Slicer (Mickle Laboratory Engineering Co., Gomshall, Surrey, England) and fractions were counted for 3H and 14C by liquid scintillation spectrometry 9. The fucose-labeled glycoproteins in the myelin fraction are dominated by a major peak migrating slightly less than halfway down the gel. The patterns of labeled myelin glycoproteins in 14- and 22-day-old rats are very similar as shown in Fig. 1A. However, the major peak was shifted slightly toward the top of the gel in the myelin from the 14-day-old rats as compared to that from the 22-day-old rats. This indicates that the newly synthesized glycoprotein from the 14-day-old rats has a slightly higher
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Fig. 1. Comparison of fucose-labeled glycoproteins in myelin from 14-day-old rats and 22-day-old rats. Electrophoresis on SDS gels was from the top of the gels shown at the left toward the anode at the right. The gels were divided into 3-mm fractions for determination of radioactivity. A: open circles, 14-day myelin labeled with 14C; closed circles, 22-day myelin labeled with 3H. B: open circles, 22-day myelin labeled with 14C; closed circles, 22-day myelin labeled with 3H.
apparent molecular weight. The shift is small but was reproducibly demonstrated by the double label technique in which the two samples are electrophoresed on the same gel. In a number of experiments in which double label gels were run comparing myelin preparations from animals of the same age, the major peaks coincided and a shift was not apparent. An example of such a control is shown in Fig. 1B. There was also little or no shift when 22-day myelin was compared to adult myelin. In the experiments described above [14C]fucose was used to label myelin from the younger animals whereas the myelin from the older animals was labeled with [3H]fucose. When the isotopes were reversed, the [3H]fucose-labeled peak from the 14-day myelin was also shifted toward higher apparent molecular weight. This rules out the possibility that the shift is an artifact due to an isotope effect. The results described above were obtained with myelin prepared from mixed homogenates. This has the advantage that the myelin from animals of both ages is exposed to exactly the same conditions during the purification procedure. However, the same difference in electrophoretic mobility of the major radioactive glycoprotein was seen when myelin was purified separately from 14- and 22-day-old rats, respectively, and mixed just prior to electrophoresis. A higher resolution comparison of the glycoprotein peaks at 14 and 22 days is shown in Fig. 2. In this experiment, the mixed myelin preparation was extensively purified by recycling the myelin 4 additional.times through the discontinuous gradients and osmotic shocks. This was done to reduce the possibility that contaminating non-
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Fig. 2. Higher resolution comparison of partially purified glycoprotein from extra pure myelin of 14-day-old and 22-day-old rats, respectively. Myelin was extensively purified and a fraction enriched in glycoproteins was obtained as described in the text. Electrophoresis was as described for Fig. 1, but the portion of the gel containing the major peak was divided into 1-mm fractions for determination of radioactivity. Open circles, 14-day myelin labeled with 14C; closed circles, 22-day myelin labeled with 3H.
myelin glycoproteins could affect the electrophoretic pattern. Also, the glycoproteins in the myelin preparation were partially purified prior to electrophoresis by treating the myelin with chloroform-methanol (2:1, v/v) which removes the lipid and threefourths of the protein. As a result of the purification, it was possible to apply more glycoprotein and more radioactivity to the gels, permitting smaller fractions of gel to be counted and therefore giving higher resolution. Fig. 2 clearly shows the higher apparent molecular weight for the glycoprotein peak in 14-day myelin compared to 22-day myelin. It is also evident from Fig. 2 that, even with the higher resolution, the radioactivity was not divided into more than one peak at either age, although there is a shoulder of the 14-day peak in the region of the 22-day peak and vice versa. The chemical reason for the developmental difference in electrophoretic mobility remains to be determined. Although lower electrophoretic mobility on SDS gels indicates higher molecular weight for most proteins, the situation may be more complicated for glycoproteins. The mobilities of glycoproteins can be affected by the amount of SDS bound and by the charge contribution of sialic acid residues 6. It seems likely that there is a single glycoprotein (i.e. one polypeptide chain) in the myelin fraction at both ages, and that the different electrophoretic mobilities reflect a difference in the carbohydrate composition of the glycoprotein being synthesized at two ages. An alternative possibility is that there are two different glycoproteins and that the relative rates of synthesis of the two change with age. This latter possibility seems less likely, since we have not detected evidence of two bands on periodic acidSchiff stained gels. In addition, we have not seen division of the radioactivity into two peaks even when 1-mm slices were used. The shoulders on the peaks shown in Fig. 2 could be explained by two forms of a single glycoprotein differing in carbohydrate composition as well as by two distinct glycoproteins. Recent results from our laboratory have indicated that the glycoprotein in
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purified myelin fractions is closely associated with myelin in the tissue, a l t h o u g h it m a y be seletively concentrated in a location such as the oligodendroglial plasma m e m b r a n e and its region o f transition to myelin, loose myelin, or the axolemma o f myelinated axons 1,5. The data reported here clearly show that the major myelinassociated glycoprotein (or glycoproteins) which is synthesized in 14-day-old rats is different f r o m that synthesized in more mature rats. Since glycoproteins are believed to be involved in recognition or contact relationships between cells, this developmental change could play an i m p o r t a n t role in the process o f myelination. It might mediate modifications o f contact relationships between oligodendroglial and axonal membranes or between different layers o f myelin membranes. Therefore, the electrophoretic difference reported in this paper could be a valuable handle for elucidating the nature o f contact relationships occurring between membranes during the process o f myelination.
1 MATTHIEU,J. M., BRADY, R. O., AND QUARLES, R. H., Comparison of the distribution of a glyco-
2
3 4 5 6 7 8 9
protein with other proteins, Gm ganglioside, and enzyme markers in subfractions of myelin, Trans. Amer. Soc. Neurochem., 4 (1973) 124 (Abstract). NORTON, W. T., Recent developments in the investigation of purified myelin. In R. PAOLETTIAND A. N. DAVISON(Eds.), Chemistry and Brain Development, Plenum Press, New York, 1971, pp. 327-337. QUARLES,R. H., ANDBRADY,R. O., Synthesis of glycoproteins and gangliosides in developing rat brain, J. Neurochem., 18 (1971) 1809-1820. QUARLES,R. H., EVERLY,J. L., AND BRADY,R. O., Demonstration of a glycoprotein which is associated with a purified myelin fraction from rat brain, Biochem. biophys. Res. Commun., 68 (1972) 491497. QUARLES, R. H., AND EVERLY, J. L., Evidence for the close association of a glycoprotein with myelin, Fed. Proc., 32 (1973) 1489 (Abstract). SEGREST,J. P., AND JACKSON,R. L., Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In V. GINSBURG(Ed.), Complex Carbohydrates, Methods in Enzymology, Vol. 28, Academic Press, New York 1972, pp. 54-63. SHAPIRO,A. L., VINUELA,E., AND MAIZEL,J. V., Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels, Biochem. biophys. Res. Commun., 28 (1967) 815-820. WINZLER,R. J., Carbohydrates in cell surfaces. In G. H. BOURNEAND J. F. DANIELLI,Int. Rev. Cytol., Vol. 29, Academic Press, New York, 1970, pp. 77-125. ZAITLIN, M., AND HARIHARASURBRAMANIAN, V., An improvement in a procedure for counting tritium and carbon-14 in polyacrylamide gels, Analyt. Biochem., 35 (1970) 296-297.
Brain Research, 58 (1973) 506-509 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Myelin.associated glycoprotein: a developmental change RICHARD H. QUARLES, JOHN L. EVERLY AND ROSCOE O. BRADY Developmental and Metabolic Neurology Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)
(Accepted May 8th, 1973)
Glycoproteins on the plasma membranes of cells are believed to be involved in specific cell-cell interactions 8. Myelin which is thought to be derived from the plasma membrane of the oligodendrocyte has generally been considered to contain little or no glycoprotein. Recently, however, we have demonstrated a glycoprotein which is closely associated with myelin4,L In this paper, we present evidence for a developmental change in this glycoprotein which could play an important role in the process of myelination. A preliminary report of this finding has been presented 5. Sprague-Dawley rats of various ages were purchased from Taconic Farms (Germantown, New York). L-[1,5,6-aH]Fucose and L-[1-14C]fucose were obtained from New England Nuclear Corporation (Boston, Mass.). The animals were injected intracerebrally 3 with 20 #Ci of [3H]fucose (125 #Ci//~mole) or 8 #Ci of [14C]fucose (50/~Ci//zmole) and sacrificed 16 h later. Five percent homogenates of the brains from these animals were prepared in 0.32 M sucrose, and myelin was purified essentially according to the procedure of Norton and Poduslo ~. In most experiments, the myelin was purified from a mixed homogenate obtained by combining equal volumes of a [3H]fucose-labeled homogenate from animals of one age and a [14C]fucose-labeled homogenate from animals of another age. The myelin was lyophilized and dissolved in 2.5 ~ sodium dodecyl sulfate (SDS), 1 ~ sodium carbonate, and 10~ mercaptoethanol. It was dialyzed aganst 0 . 1 ~ SDS, 0.01 M sodium phosphate buffer (pH 7.2), 1.6 M urea, and 0.05 ~ dithiothreitol. The samples were electrophoresed on 5 ~ SDS-polyacrylamide gels (11 cm × 6 mm) prepared according to Shapiro et aLL After electrophoresis the gels were fractionated on a Mickle Gel Slicer (Mickle Laboratory Engineering Co., Gomshall, Surrey, England) and fractions were counted for 3H and 14C by liquid scintillation spectrometry 9. The fucose-labeled glycoproteins in the myelin fraction are dominated by a major peak migrating slightly less than halfway down the gel. The patterns of labeled myelin glycoproteins in 14- and 22-day-old rats are very similar as shown in Fig. 1A. However, the major peak was shifted slightly toward the top of the gel in the myelin from the 14-day-old rats as compared to that from the 22-day-old rats. This indicates that the newly synthesized glycoprotein from the 14-day-old rats has a slightly higher
507
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3'
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l
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Fig. 1. Comparison of fucose-labeled glycoproteins in myelin from 14-day-old rats and 22-day-old rats. Electrophoresis on SDS gels was from the top of the gels shown at the left toward the anode at the right. The gels were divided into 3-mm fractions for determination of radioactivity. A: open circles, 14-day myelin labeled with 14C; closed circles, 22-day myelin labeled with 3H. B: open circles, 22-day myelin labeled with 14C; closed circles, 22-day myelin labeled with 3H.
apparent molecular weight. The shift is small but was reproducibly demonstrated by the double label technique in which the two samples are electrophoresed on the same gel. In a number of experiments in which double label gels were run comparing myelin preparations from animals of the same age, the major peaks coincided and a shift was not apparent. An example of such a control is shown in Fig. 1B. There was also little or no shift when 22-day myelin was compared to adult myelin. In the experiments described above [14C]fucose was used to label myelin from the younger animals whereas the myelin from the older animals was labeled with [3H]fucose. When the isotopes were reversed, the [3H]fucose-labeled peak from the 14-day myelin was also shifted toward higher apparent molecular weight. This rules out the possibility that the shift is an artifact due to an isotope effect. The results described above were obtained with myelin prepared from mixed homogenates. This has the advantage that the myelin from animals of both ages is exposed to exactly the same conditions during the purification procedure. However, the same difference in electrophoretic mobility of the major radioactive glycoprotein was seen when myelin was purified separately from 14- and 22-day-old rats, respectively, and mixed just prior to electrophoresis. A higher resolution comparison of the glycoprotein peaks at 14 and 22 days is shown in Fig. 2. In this experiment, the mixed myelin preparation was extensively purified by recycling the myelin 4 additional.times through the discontinuous gradients and osmotic shocks. This was done to reduce the possibility that contaminating non-
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Fig. 2. Higher resolution comparison of partially purified glycoprotein from extra pure myelin of 14-day-old and 22-day-old rats, respectively. Myelin was extensively purified and a fraction enriched in glycoproteins was obtained as described in the text. Electrophoresis was as described for Fig. 1, but the portion of the gel containing the major peak was divided into 1-mm fractions for determination of radioactivity. Open circles, 14-day myelin labeled with 14C; closed circles, 22-day myelin labeled with 3H.
myelin glycoproteins could affect the electrophoretic pattern. Also, the glycoproteins in the myelin preparation were partially purified prior to electrophoresis by treating the myelin with chloroform-methanol (2:1, v/v) which removes the lipid and threefourths of the protein. As a result of the purification, it was possible to apply more glycoprotein and more radioactivity to the gels, permitting smaller fractions of gel to be counted and therefore giving higher resolution. Fig. 2 clearly shows the higher apparent molecular weight for the glycoprotein peak in 14-day myelin compared to 22-day myelin. It is also evident from Fig. 2 that, even with the higher resolution, the radioactivity was not divided into more than one peak at either age, although there is a shoulder of the 14-day peak in the region of the 22-day peak and vice versa. The chemical reason for the developmental difference in electrophoretic mobility remains to be determined. Although lower electrophoretic mobility on SDS gels indicates higher molecular weight for most proteins, the situation may be more complicated for glycoproteins. The mobilities of glycoproteins can be affected by the amount of SDS bound and by the charge contribution of sialic acid residues 6. It seems likely that there is a single glycoprotein (i.e. one polypeptide chain) in the myelin fraction at both ages, and that the different electrophoretic mobilities reflect a difference in the carbohydrate composition of the glycoprotein being synthesized at two ages. An alternative possibility is that there are two different glycoproteins and that the relative rates of synthesis of the two change with age. This latter possibility seems less likely, since we have not detected evidence of two bands on periodic acidSchiff stained gels. In addition, we have not seen division of the radioactivity into two peaks even when 1-mm slices were used. The shoulders on the peaks shown in Fig. 2 could be explained by two forms of a single glycoprotein differing in carbohydrate composition as well as by two distinct glycoproteins. Recent results from our laboratory have indicated that the glycoprotein in
SHORT COMMUNICATIONS
509
purified myelin fractions is closely associated with myelin in the tissue, a l t h o u g h it m a y be seletively concentrated in a location such as the oligodendroglial plasma m e m b r a n e and its region o f transition to myelin, loose myelin, or the axolemma o f myelinated axons 1,5. The data reported here clearly show that the major myelinassociated glycoprotein (or glycoproteins) which is synthesized in 14-day-old rats is different f r o m that synthesized in more mature rats. Since glycoproteins are believed to be involved in recognition or contact relationships between cells, this developmental change could play an i m p o r t a n t role in the process o f myelination. It might mediate modifications o f contact relationships between oligodendroglial and axonal membranes or between different layers o f myelin membranes. Therefore, the electrophoretic difference reported in this paper could be a valuable handle for elucidating the nature o f contact relationships occurring between membranes during the process o f myelination.
1 MATTHIEU,J. M., BRADY, R. O., AND QUARLES, R. H., Comparison of the distribution of a glyco-
2
3 4 5 6 7 8 9
protein with other proteins, Gm ganglioside, and enzyme markers in subfractions of myelin, Trans. Amer. Soc. Neurochem., 4 (1973) 124 (Abstract). NORTON, W. T., Recent developments in the investigation of purified myelin. In R. PAOLETTIAND A. N. DAVISON(Eds.), Chemistry and Brain Development, Plenum Press, New York, 1971, pp. 327-337. QUARLES,R. H., ANDBRADY,R. O., Synthesis of glycoproteins and gangliosides in developing rat brain, J. Neurochem., 18 (1971) 1809-1820. QUARLES,R. H., EVERLY,J. L., AND BRADY,R. O., Demonstration of a glycoprotein which is associated with a purified myelin fraction from rat brain, Biochem. biophys. Res. Commun., 68 (1972) 491497. QUARLES, R. H., AND EVERLY, J. L., Evidence for the close association of a glycoprotein with myelin, Fed. Proc., 32 (1973) 1489 (Abstract). SEGREST,J. P., AND JACKSON,R. L., Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In V. GINSBURG(Ed.), Complex Carbohydrates, Methods in Enzymology, Vol. 28, Academic Press, New York 1972, pp. 54-63. SHAPIRO,A. L., VINUELA,E., AND MAIZEL,J. V., Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels, Biochem. biophys. Res. Commun., 28 (1967) 815-820. WINZLER,R. J., Carbohydrates in cell surfaces. In G. H. BOURNEAND J. F. DANIELLI,Int. Rev. Cytol., Vol. 29, Academic Press, New York, 1970, pp. 77-125. ZAITLIN, M., AND HARIHARASURBRAMANIAN, V., An improvement in a procedure for counting tritium and carbon-14 in polyacrylamide gels, Analyt. Biochem., 35 (1970) 296-297.