Skip to main content
SpringerLink
Log in

Cleavage of the PO glycoprotein of the rat peripheral nerve myelin: Tentative identification of cleavage site and evidence for the precursor-product relationship

  • Original Articles
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37°C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24kDa protein with a concomitant decrease of PO protein. The conversion of PO into 24kDa protein was blocked by heating isolated myelin at 100°C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24kDa protein. Similarly, addition of CaCl2 to isolated myelin did not accentuate the formation of 24kDa protein suggesting that the conversion of PO into 24kDa protein may not be due to Ca2+ activated protease. It is postulated that the formation of 24kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24kDa protein was purified and characterized. The N-terminal sequence of 1–17 amino acid residues of 24kDa protein was identical to PO. 24kDa protein was immunostained and immunoprecipitated with anti-PO antiserum indicating the immunological similarities between PO and 24kDa protein. Labeling of 24kDa protein with [35S]methionine provided evidence that PO may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24kDa protein was phosphorylated, glycosylated and acylated like PO. Phosphorylation of 24kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24kDa protein. These results suggested that serine residue phosphorylated by protein kinase C may be located in amino acid residues 1-168. 24kDa protein was stained with periodic Schiff reagent. In addition, 24kDa protein was fucosylated and the fucosylation of 24kDa protein was inhibited (70%) by tunicamycin, providing evidence that it is N-glycosylated. Recently, it was demonstrated that both PO and 24kDa protein were fatty acylated with [3H]palmitic acid in the nerve slices and fatty acids are covalently linked to these proteins (Agrawal, H.C. and Agrawal, D. 1989, Biochem. J. 263:173–177). The time course of inhibition of acylation by cycloheximide of 24kDa protein was identical to PO. Cycloheximide inhibited acylation of PO and 24kDa protein by 61% and 58% respectively, whereas, monensin had little affect on the fatty acylation of these proteins. Less [3H]palmitic acid and14C-amino acids were incorporated into 24kDa protein when compared to PO between 5–30 min after incubation of the nerve slices. However, more radioactivity was incorporated into 24kDa protein after 60 min when compared to PO under identical conditions. These results provided evidence of a precursor-product relationship between PO and 24kDa protein. Therefore, PO may be cleaved into 24kDa protein in the myelin membrane following its acylation and glycosylation in the Schwann cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Shuman, S., Hardy, M., and Pleasure, D. 1983. Peripheral nervous system myelin and Schwann cell glycoproteins: Identification by lectin binding and partial purification of a peripheral nervous system myelin-specific 170,000 molecular weight glycoprotein. J. Neurochem. 41:1277–1285.

    PubMed  Google Scholar 

  2. Sternberger, N. H., Quarles, R. H., Itoyama, Y., and Webster, H. deF. 1979. Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat. Proc. Natl. Acad. Sci., USA, 76:1510–1514.

    Google Scholar 

  3. Figlewicz, D. A., Quarles, R. H., Johnson, D., Barbarash, G. R., and Sternberger, N. H. 1981. Biochemical demonstration of the myelin-associated glycoprotein in the peripheral nervous system. J. Neurochem. 37:749–758.

    PubMed  Google Scholar 

  4. Agrawal, H. C., Noronha, A. B., Agrawal, D., and Quarles, R. H. 1990. The myelin associated glycoprotein is phosphorylated in the peripheral nervous system. Biochem. Biophys. Res. Commun. 169:953–958.

    PubMed  Google Scholar 

  5. Sprinkle, T. J. 1989. 2′,3′-cyclic Nucleotide-3′-Phosphodiesterase, An Oligodendrocyte-Schwann cell and Myelin-Associated Enzyme of the Nervous System. CRC: Critical Reviews in Neurobiology 4:235–301.

    Google Scholar 

  6. Agrawal, H. C., Sprinkle, T. J., and Agrawal, D. 1990. 2′,3′ cyclicnucleotide-3′-phosphodiesterase in peripheral nerve myelin is phosphorylated by a phorbol ester-sensitive kinase. Biochem. Biophys. Res. Commun. 170:817–823.

    PubMed  Google Scholar 

  7. Greenfield, S., Brostoff, S., Eylar, E. H., and Morell, P. 1973. Protein composition of myelin of the peripheral nervous system. J. Neurochem. 20:1207–1216.

    PubMed  Google Scholar 

  8. Wood, J. G., and Dawson, R. M. C. 1973. A major myelin glycoprotein of sciatic nerve. J. Neurochem. 21:717–719.

    PubMed  Google Scholar 

  9. Quarles, R. H., Everly, J. L., and Brady, R. O. 1973. Evidence that the major protein in rat sciatic nerve is a glycoprotein. J. Neurochem. 21:329–334.

    PubMed  Google Scholar 

  10. Greenfield, S., Weise, M. J., Gantt, G. Hogan, E. L., and Brostoff, S. W. 1982. Basic proteins of rodent peripheral nerve myelin: Immunochemical identification of the 21.5K, 18.5K, 17K, 14K and P2 protein. J. Neurochem. 39:1278–1282.

    PubMed  Google Scholar 

  11. Gilbert, W.R., Garwood, M. M., Agrawal, D., Schmidt, R. E., and Agrawal, H. C. 1982. Immunoblot identification of phosphorylated basic proteins of rat and rabbit CNS and PNS myelin: Evidence for four phosphorylated basic proteins and P2 in rat PNS myelin. Neurochem. Res. 7:1495–1506.

    PubMed  Google Scholar 

  12. Ishaque, A., Roomi, M. W., Szymanska, I., Kowalski, S., and Eylar, E. H. 1980. The P0 glycoprotein of peripheral nerve myelin. Can. J. Biochem. 58:913–921.

    PubMed  Google Scholar 

  13. Smith, M. E., and Perret, V. 1986. Immunological non-identity of 19K protein and TPO in peripheral nerve myelin. J. Neurochem. 47:924–929.

    PubMed  Google Scholar 

  14. Trapp, B. O., McIntyre, L. J., Quarles, R. H., Sternberger, N. H., and Webster, H. deF. 1979. Immunocytochemical localization of rat peripheral nervous system myelin proteins: P2 protein is not a component of all peripheral nervous system myelin sheaths. Proc. Natl. Acad. Sci., USA, 76:3552–3556.

    Google Scholar 

  15. Trapp, B. O., Itoyama, Y., Sternberger, N. H., Quarles, R. H., and Webster, H. deF. 1981. Immunocytochemical localization of PO protein in Golgi complex membranes and myelin of developing rat Schwann cells. J. Cell Biol. 90:1–6.

    PubMed  Google Scholar 

  16. Poduslo, J. F., and Yao, J. K. 1985. Association and release of the major intrinsic membrane glycoprotein from peripheral nerve myelin. Biochem. J. 228:43–54.

    PubMed  Google Scholar 

  17. Matthieu, J. M., Everly, J. L., Brady, R. O., and Quarles, R. H. 1975. [35S] Sulfate incorporation into myelin glycoproteins. II. Peripheral nervous tissue. Biochim. Biophys. Acta 392:167–174.

    PubMed  Google Scholar 

  18. Kitamura, K., Sakamoto, Y., Suzuki, M., and Uyemura, K. 1981. Microheterogeneity of a carbohydrate in PO protein from bovine peripheral nerve myelin,in Proceedings of the 6th International Symposium on Glycoconjugates (Yamakawa, T., Osawa T. and Handa S., eds), pp. 273–274. Japan Scientific Societies Press, Tokyo.

    Google Scholar 

  19. Singh, H., and Spritz, N. 1976. Protein kinase associated with peripheral nerve myelin. I. Phosphorylation of endogenous myelin proteins and exogenous substrates. Biochim. Biophys. Acta 448:325–337.

    PubMed  Google Scholar 

  20. Petrali, E. H., and Sulakhe, P. V. 1979. Calcium ion stimulated endogenous protein kinase catalyzed phosphorylation of peripheral nerve myelin proteins. Can. J. Physiol. Pharmacol. 57:1200–1204.

    PubMed  Google Scholar 

  21. Wiggins, R. C., and Morell, P. 1980. Phosphorylation and fucosylation of myelin proteinin vitro by sciatic nerve from developing rats. J. Neurochem. 34:627–634.

    PubMed  Google Scholar 

  22. Toews, A. D., Fischer, H. R., Goodrum, J. F., Windes, S., and Morell, P. 1987. Metabolism of phosphate and sulfate groups modifying the PO protein of peripheral nervous system myelin. J. Neurochem 48:883–887.

    PubMed  Google Scholar 

  23. Brunden, J. R., and Poduslo, J. F. 1987. A phorbol ester-sensitive kinase catalyzes the phosphorylation of PO glycoprotein in myelin. J. Neurochem. 49:1863–1872.

    PubMed  Google Scholar 

  24. Agrawal, H. C., and Agrawal, D. 1989. Tumor promoters accentuate phosphorylation of PO: Evidence for the presence of protein kinase C in purified PNS myelin. Neurochem. Res. 14:409–413.

    PubMed  Google Scholar 

  25. Agrawal, H. C., Schmidt, R. E., and Agrawal, D. 1983. In vivo incorporation of [3H] palmitic acid into PO protein, the major intrinsic protein of rat sciatic nerve myelin. J. Biol. Chem. 258:6556–6560.

    PubMed  Google Scholar 

  26. Sakamoto, J., Kitamura, K., Yoshimura, K., Nishijima, T., and Uyemura, K. 1986. Fatty acid linked peptide from bovine PO protein in peripheral nerve myelin. Biomedical. Res. 7:261–266

    Google Scholar 

  27. Agrawal, H. C., and Agrawal, D. 1989. Effect of cycloheximide on palmitylation of PO protein of the peripheral nervous system myelin. Biochem. J. 263:173–177.

    PubMed  Google Scholar 

  28. Bizzozero, O. A., Odykirk, T. S., McGarry, J. F., and Lees, M. B. 1989. Separation of the major proteins of central and peripheral nervous system myelin using reversed-phase high performance liquid chromatography. Anal. Biochem. 180:56–59.

    Google Scholar 

  29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265–275.

    PubMed  Google Scholar 

  30. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227:680–685.

    Google Scholar 

  31. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl. Acad. Sci., USA, 76:4350–4354.

    Google Scholar 

  32. Nunn, D. J., Leblanc, A. C., and Mezei, C. 1987. A 42K protein of chick sciatic nerve is immunologically related to PO protein of peripheral nerve myelin. Neurochem. Res. 12:377–384.

    PubMed  Google Scholar 

  33. Agrawal, H. C., Agrawal, D., and Jenkins, R. J. 1986. Immunochemical evidence of phosphorylation of a new 23K basic protein in rat brain myelin. Neurochem. Res. 11:375–382.

    PubMed  Google Scholar 

  34. Goldman, B. M., and Blobel, G. 1978. Biogenesis of peroxisomes: Intracellular site of synthesis of synthesis of catalase and uricase. Proc. Natl. Acad. Sci., USA, 75:5066–5070.

    Google Scholar 

  35. Agrawal, H. C., Sprinkle, T. J., and Agrawal, D. 1990. 2′,3′ cyclic nucleotide-3′-phosphodiesterase in the CNS is fatty acylated by thioester linkage. J. Biol. Chem. 265:11849–11853.

    PubMed  Google Scholar 

  36. Agrawal, H. C., Randle, C. L., Agrawal, D. 1981. In vivo phosphorylation of two myelin basic proteins of developing rabbit brain. J. Biol. Chem. 256:12243–12246.

    PubMed  Google Scholar 

  37. Fairbanks, G., Steck, T. L., and Wallach, D. F. 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617.

    PubMed  Google Scholar 

  38. Sato, S., Brady, R. O., and Quarles, R. H. 1982. Susceptibility of the myelin-associated glycoprotein and basic protein to a neutral protease in highly purified myelin from human and rat brain. J. Neurochem. 39:97–105.

    PubMed  Google Scholar 

  39. Sato, S., Yanagisawa, K., and Miyatake, T. 1986. Conversion of myelin associated glycoprotein (MAG) to a smaller derivative by calcium activated neutral protease (CANP)-like enzyme in myelin and inhibition by E-64 analogue. Neurochem. Res. 9:629–635.

    Google Scholar 

  40. Yanagisawa, K., Sato, S., O'Shannessy, O. J., Quarles, R. H., Suzuki, K., and Miyatake, T. 1988. Myelin-associated Calpain II. J. Neurochem. 51:803–807.

    PubMed  Google Scholar 

  41. Lemke, G., and Axel, R. 1985. Isolation and sequence of cDNA encoding the major structural protein of peripheral myelin. Cell, 40:501–508.

    PubMed  Google Scholar 

  42. Sakamoto, Y., Kitamura, K., Yoshimura, K., Nishijima, T., and Uyemura, K. 1987. Complete amino acid sequence of PO protein in bovine peripheral nerve myelin. J. Biol. Chem. 262:4208–4214.

    PubMed  Google Scholar 

  43. Kitamura, K., Suzuki, A., Suzuki, M., and Uyemura, K. 1979. Amino acid sequence of the glycopeptide derived from a major glycoprotein in bovine peripheral nerve myelin. FEBS. Lett. 100:67–70.

    PubMed  Google Scholar 

  44. Agrawal, H. C., Randle, C. L., and Agrawal, D. 1982. In vivo acylation of rat brain myelin proteolipid protein. J. Biol. Chem. 257:4588–4592.

    PubMed  Google Scholar 

  45. Rapaport, R. N., Benjamins, J. A. and Skoff, R. P. 1982. Effects of monensin on assembly of PO protein. J. Neurochem. 39:1101–1110.

    PubMed  Google Scholar 

  46. Benjamins, J. A., and Morell, P. 1978. Proteins of myelin and their metabolism. Neurochem. Res. 3:137–174.

    PubMed  Google Scholar 

  47. Cammer, W., Sirota, S. R., and Norton, W. T. 1981. The effect of reducing agents on the apparent molecular weight of the myelin PO protein and the possible identity of the PO and “Y” proteins. J. Neurochem. 34:404–409.

    Google Scholar 

  48. Wood, J. G., and McLaughlin, B. L. 1975. The visualization of concavalin-A binding sites in the intraperiod line of rat sciatic nerve myelin. J. Neurochem. 24:233–235.

    PubMed  Google Scholar 

  49. Kirschner, D. A., and Ganser, A. L. 1980. Compact myelin exists in the absence of basic protein in the Shiverer mutant mouse. Nature 238:207–210.

    Google Scholar 

  50. Rosenbluth, J. 1980. Peripheral myelin in the mouse mutant Shiverer. J. Comp. Neurol. 193:729–739.

    PubMed  Google Scholar 

  51. Mikoshiba, K., Kohsaka, S., Takamatsu, K., and Tsukada, Y. 1981. Neurochemical and morphological studies on the myelin of peripheral nervous system from Shiverer mutant mice. Absence of basic proteins common to central nervous system. Brain Res. 204:455–460.

    PubMed  Google Scholar 

  52. Martenson, R. E. 1980. Myelin basic protein: What does it do? In Biochemistry of Brain (S. Kumar, ed.) Pergamon Press, pp. 49–80.

  53. Benjamins, J. A., Morell, P., Hartman, B. K. and Agrawal, H. C. 1984. Central nervous system myelin. Pages 361–414,in Handbook of Neurochemistry A. Lajtha, (ed.), Vol. 7 Plenum Press, New York.

    Google Scholar 

  54. Martenson, R. E., Law, M. J., and Deibler, G. E. 1983. Identification of multiple in vivo phosphorylation sites in rabbit myelin basic protein. J. Biol. Chem. 258:930–937.

    PubMed  Google Scholar 

  55. Scott Turner, S., Chow, C-H. Jen, Kibler, R. F., and Kuo, J. F. 1982. Basic protein in brain myelin is phosphorylated by endogenous phospholipid-sensitive Ca2+ dependent protein kinase. J. Neurochem. 39:1397–1404.

    PubMed  Google Scholar 

  56. Vartanian, T., Szuchet, S., Dawson, D., and Campagnoni, A. T. 1986. Oligodendrocyte substrates adhesion modulates expression of adenylate cyclase-linked receptors. Science 234:1395–1398.

    PubMed  Google Scholar 

  57. Marks, N. Grynbaum, A. and Lajtha, A. 1976. The breakdown of myelin-bound proteins by intra- and extracellular proteases. Neurochem. Res. 1:93–111.

    Google Scholar 

  58. Fishman, M. A., Trotter, J. L., and Agrawal, H. C. 1977. Selective loss of myelin proteins during autolysis. Neurochem. Res. 2:247–257.

    Google Scholar 

  59. Cammer, W., Bloom, B. R., Norton, W. T. and Gordon, S. 1978. Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: A possible mechanism of inflammatory demyelination. Proc. Natl. Acad. Sci. USA, 75:1554–1558.

    PubMed  Google Scholar 

  60. Banik, N. L. 1979. The degradation of myelin basic protein by serum protease in experimental allergic encephalomyelitis and control rats. Neurosci. Lett. 11:307–312.

    PubMed  Google Scholar 

  61. Whitaker, J. N., and Seyer, J. M. 1981. The influence of pH on the degradation of bovine myelin basic protein by bovine brain cathepsin D. Biochim. Biophys. Acta 661:334–341.

    PubMed  Google Scholar 

  62. Singh, I., and Singh, A. K. 1983. Degradation of myelin proteins by brain endogenous neutral protease. Neurosci. Lett. 39:77–82.

    PubMed  Google Scholar 

  63. Berlet, H. H. Ilzenhöfer, H., and Schultz, G. 1984. Cleavage of myelin basic protein by neutral protease activity of human white matter and myelin. J. Neurochem. 43:627–633.

    PubMed  Google Scholar 

  64. Deibler, G. E., Boyd, L. F., and Kies, M. W. 1984. Enzymatic and nonenzymatic degradation of myelin basic protein. Neurochem. Res. 9:1371–1385.

    PubMed  Google Scholar 

  65. Banik, N. L., Happel, R. D. Sostek, M. B., Chiu, F. C., and Hogan, E. L. 1987. Ca2+ mediated degradation of central nervous system (CNS) proteins: Topographic and species variation. Metabolic Brain Res 2:117–126.

    Google Scholar 

  66. Chantry, A., Gregson, N. A., and Glynn, P. 1989. A novel metalloproteinase associated with brain myelin membranes. Isolation and characterization. J. Biol. Chem. 264:21603–2607.

    PubMed  Google Scholar 

Download references

Authors
  1. Harish C. Agrawal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daya Agrawal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arnold W. Strauss
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Agrawal, H.C., Agrawal, D. & Strauss, A.W. Cleavage of the PO glycoprotein of the rat peripheral nerve myelin: Tentative identification of cleavage site and evidence for the precursor-product relationship. Neurochem Res 15, 993–1001 (1990). https://doi.org/10.1007/BF00965745

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00965745

Key Words

Search

Navigation