Abstract
Proteolipids were isolated from 20 day old normal andjimpy mouse brain by extraction into chloroform-methanol (2∶1, w/v), delipidated by size-exclusion HPLC, and analyzed by SDS-PAGE, Western blots, amino acid analyses, and N-terminal sequencing. SDS-PAGE showed that a major proteolipid fromjimpy mouse brain had an apparent molecular weight of ∼23 kDa, intermediate to that of PLP and DM-20 from normal mouse brain. Western blots with 3 different antibodies which recognize residues 200–224, 116–150, and 270–276 respectively recognized immunoreactive material in normal andjimpy PLP. Since antibody reactive with 270–276 did not recognizejimpy PLP, an altered C-terminus of thejimpy protein is suggested. These results demonstrated that a PLP can be partially purified fromjimpy mouse brain. Amino acid analyses failed to show the predicted increase in cysteinyl residues (predicted from cDNA) injimpy PLP. However, whenjimpy brain proteolipids were subjected to N-terminal sequencing, Gly, Leu, Leu, Gly the first four amino acids of PLP were detected. Thus, the partial purification of a proteolipid fromjimpy mouse brain, whose characteristics (apparent molecular weight, immunoreactivity, N-terminal sequence and relative net charge) strongly suggested that PLP of altered size is present injimpy mouse brain.
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Abbreviations
- BCIP:
-
5-bromo-4-chloro-3-indolyl phosphate toluidine salt
- MBP:
-
myelin basic protein
- NBT:
-
ρ-nitro blue tetrazolium chloride
- PITC:
-
phenylisothiocyanate
- PLP:
-
myelin proteolipid protein
- PVDF:
-
polyvinylidene difluoride
- SDS-PAGE:
-
sodium dodecyl sulfate polyacrylamide gel electrophoresis
References
Bartlett, G. R. 1959. Phosphorous assay in column chromatography. J. Biol. Chem. 234:466–468.
Bartlett, W. J., Knapp, P. E., and Skoff, R. P. 1988. Glial Conditioned medium enablesjimpy oligodendrocytes to express properties of normal oligodendrocytes: Production of myelin antigens and membranes. Glial 1:253–259.
Boggs, J. M., Moscarello, M. A., and Papahadjopoulos, D. 1982. Structural organization of myelin—role of lipid-protein interactions determined in model systems,in Jost, P., and Griffith, O. H. (eds.), Lipid-Protein Interactions pp. 1–51. John Wiley & Sons, New York.
Campagnoni, A. T., and Macklin, W. B. 1988. Cellular and molecular aspects of myelin protein gene expression. Mol. Neurobiol. 2:41–89.
Cochary, E. F., Bizzozero, O. A., Sapirstein, V. S., Nolan, C. E., and Fischer, I. 1990. Presence of the plasma membrane proteolipid (plasmolipin) in myelin. J. Neurochem. 55:602–610.
Dautigny, A., Mattei, M.-G., Morello, D., Alliel, P. M., Pham-Dinh, D., Amar, L., Arnaud, D., Simon, D., Mattei, J.-F., Guenet, J.-L., Jollès, P., and Avner, P. 1986. The structural gene coding for myelin-associated proteolipid protein is mutated injimpy mice. Nature 321:867–869.
Duncan, I. D., Skoff, R. P., and Colman, D., eds. 1990. Annals of The New York Academy of Sciences, Vol. 605: Myelination and Dysmyelination. The New York Academy of Sciences, New York.
Fannon, A. M. 1991. Myelinogenesis in the absence of normal myelin proteolipids: A study using the dysmyelinating mutantjimpy mouse. Ph.D. Thesis, University of Toronto.
Fannon, A. M., and Moscarello, M. A. 1990. Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in thejimpy mouse. Biochem. J. 268:105–110.
Fischer, I., and Sapirstein, V. S. 1986. Characterization and biosynthesis of the plasma membrane proteolipid protein in neural tissue. J. Neurochem. 47:232–238.
Hampson, D. R., and Poduslo, S. E. 1986. Purification of proteolipid protein and production of specific antiserum. J. Neuroimmunol. 11:117–129.
Hoppe, J., and Sebald, W. 1984. The proton conducting Fo-part of bacterial ATP synthases. Biophys. Biochem. Acta. 768:1–27.
Hudson, L. D., Berndt, J. A., Puckett, C., Kozak, C. A., and Lazzarini, R. A. 1987. Aberrant splicing of proteolipid protein mRNA in the dysmyelinatingjimpy mutant mouse. Proc. Natl. Acad. Scci. USA 84:1454–1458.
Ikenaka, K., Kagawa, T., and Mikoshiba, K. 1992. Selective expression of DM-20, an alternatively spliced myelin proteolipid protein gene product, in developing nervous system and in non glial cells. J. Neurochem. 58:2248.
Knapp, P. E., and Skoff, R. P. 1987. A defect in the cell cycle of neuroglia in the myelin deficientjimpy mouse. Dev. Brain Res. 35:301–306.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.
Lepage, P., Helynck, G., Chu, J.-Y., Luu, b., Sorokine, O., Trifilieff, E., and Van Dorsselaer, A. 1986. Purification and characterization of minor brain proteolipids: Use of fast atom bombardment-mass spectrometry for peptide sequencing. Biochimie 68:669–686.
Macklin, W. B., Gardinier, M. V., King, K. D., and Kampf, K. 1987. An AG→GG transition at a splice site in the myelin proteolipid protein gene injimpy mice results in the removal of an exon. FEBS Lett. 223:417–421.
Merril, L. R., Goldman, D., Sedman, S. A., and Edert, M. H. 1981. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437–1438.
Morello, D., Dautigny, A., Pham-Dinh, D., and Jollès, P. 1986. Myelin proteolipid protein (PLP and DM-20) transcripts are deleted injimpy mutant mice. EMBO J. 5:3489–3493.
Nadon, N. L., Duncan, I. D., and Hudson, L. D. 1990. A point mutation in the proteolipid protein gene of the ‘shaking pup’ interrupts oligodendrocyte development. Devel. 110:529–537.
Nave, K.-A., Lai, C., Bloom, F. E., and Milner, R. J. 1986.jimpy mutant mouse: A 74-base deletion in the mRNA for myelin proteolipid protein and evidence for a primary defect in RNA splicing. Proc. Natl. Acad. Sci. USA 83:9264–9268.
Nave, K.-A., Lai, C., Bloom, F. E., and Milner, R. J. 1987. A single nucleotide difference in the gene for myelin proteolipid protein defines thejimpy mutation in mouse. J. Neurochem. 46:1873–1877.
Norton, W. T., and Poduslo, S. 1973. Myelination in rat brain: Method of myelin isolation. J. Neurochem. 21:749–757.
Peterson, G. L. 1979. Review of the folin phenol protein quantiation method of Lowry, Rosbrough, Farr and Randall. Anal. Biochem. 100:201–220.
Puckett, C., Hudson, L. D., Ono, K., Friedrich, V. Benecke, J., Dubois-Dalq, M., and Lazzarini, R. A. 1987. Myelin-specific proteolipid protein is expressed in myelinating Schwann cells but is not incorporated into myelin sheaths. J. Neurosci. Res. 18:511–518.
Roach, A., Takahashi, N., Pravtcheva, D., Duddle, F., and Hood, L. 1985. Chromosomal mapping of mouse myelin basic protein gene and structure and transcription of the partially deleted gene inshiverer mutant mice. Cell 42:149–155.
Roussel, G., Neskovic, N. M., Trifilieff, E., Artault, J.-C., and Nussbaum, J.-L. 1987. Arrest of proteolipid transport through the golgi apparatus injimpy brain. J. Neurocytol. 16:195–204.
Simons, R., Alon, N., and Riordan, J. R. 1987. Human myelin DM-20 proteolipid protein deletion defined by cDNA sequence. Biochem. Biophys. Res. Comm. 146:666–671.
Skalidis, G., Trifilieff, E., and Luu, B. 1986. Selective extraction of the DM-20 brain proteolipid. J. Neurochem. 46:297–299.
Stoffel, W., Giersiefen, H., Hillen, H., Schroeder, W., and Tunggal, B. 1985. Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved. Biol. Chem. Hoppe-Seyler 366:627–635.
Yanagisawa, K., and Quarles, R. H. 1986.jimpy mice: Quantitation of myelin-associated glycoprotein and other proteins. J. Neurochem. 47:322–325.
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Special issue dedicated to Dr. Marjorie B. Lees.
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Fannon, A.M., Mastronardi, F.G. & Moscarello, M.A. Isolation and identification of proteolipid proteins injimpy mouse brain. Neurochem Res 19, 1005–1012 (1994). https://doi.org/10.1007/BF00968710
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DOI: https://doi.org/10.1007/BF00968710