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. 1993 Dec 2;123(6):1463–1473. doi: 10.1083/jcb.123.6.1463

440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons

PMCID: PMC2290908  PMID: 8253844

Abstract

440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible alpha- helix or beta sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination.

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Selected References

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  1. Bartsch U., Kirchhoff F., Schachner M. Immunohistological localization of the adhesion molecules L1, N-CAM, and MAG in the developing and adult optic nerve of mice. J Comp Neurol. 1989 Jun 15;284(3):451–462. doi: 10.1002/cne.902840310. [DOI] [PubMed] [Google Scholar]
  2. Bennett V. Ankyrins. Adaptors between diverse plasma membrane proteins and the cytoplasm. J Biol Chem. 1992 May 5;267(13):8703–8706. [PubMed] [Google Scholar]
  3. Bennett V. Purification of an active proteolytic fragment of the membrane attachment site for human erythrocyte spectrin. J Biol Chem. 1978 Apr 10;253(7):2292–2299. [PubMed] [Google Scholar]
  4. Binder L. I., Frankfurter A., Rebhun L. I. The distribution of tau in the mammalian central nervous system. J Cell Biol. 1985 Oct;101(4):1371–1378. doi: 10.1083/jcb.101.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bird T. D., Farrell D. F., Sumi S. M. Brain lipid composition of the shiverer mouse: (genetic defect in myelin development). J Neurochem. 1978 Jul;31(1):387–391. doi: 10.1111/j.1471-4159.1978.tb12479.x. [DOI] [PubMed] [Google Scholar]
  6. Black J. A., Foster R. E., Waxman S. G. Rat optic nerve: freeze-fracture studies during development of myelinated axons. Brain Res. 1982 Oct 28;250(1):1–20. doi: 10.1016/0006-8993(82)90948-9. [DOI] [PubMed] [Google Scholar]
  7. Blaugrund E., Sharma S., Schwartz M. L1 immunoreactivity in the developing fish visual system. Brain Res. 1992 Mar 6;574(1-2):244–250. doi: 10.1016/0006-8993(92)90823-r. [DOI] [PubMed] [Google Scholar]
  8. Bloom G. S., Luca F. C., Vallee R. B. Microtubule-associated protein 1B: identification of a major component of the neuronal cytoskeleton. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5404–5408. doi: 10.1073/pnas.82.16.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Y. H., Yang J. T. A new approach to the calculation of secondary structures of globular proteins by optical rotatory dispersion and circular dichroism. Biochem Biophys Res Commun. 1971 Sep 17;44(6):1285–1291. doi: 10.1016/s0006-291x(71)80225-5. [DOI] [PubMed] [Google Scholar]
  10. Chernoff G. F. Shiverer: an autosomal recessive mutant mouse with myelin deficiency. J Hered. 1981 Mar-Apr;72(2):128–128. doi: 10.1093/oxfordjournals.jhered.a109442. [DOI] [PubMed] [Google Scholar]
  11. Correas I., Díaz-Nido J., Avila J. Microtubule-associated protein tau is phosphorylated by protein kinase C on its tubulin binding domain. J Biol Chem. 1992 Aug 5;267(22):15721–15728. [PubMed] [Google Scholar]
  12. Davis J. Q., Bennett V. Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel. J Biol Chem. 1984 Nov 10;259(21):13550–13559. [PubMed] [Google Scholar]
  13. Davis J. Q., Bennett V. Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain. J Biol Chem. 1984 Feb 10;259(3):1874–1881. [PubMed] [Google Scholar]
  14. Davis J. Q., Bennett V. The anion exchanger and Na+K(+)-ATPase interact with distinct sites on ankyrin in in vitro assays. J Biol Chem. 1990 Oct 5;265(28):17252–17256. [PubMed] [Google Scholar]
  15. Davis J. Q., McLaughlin T., Bennett V. Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain. J Cell Biol. 1993 Apr;121(1):121–133. doi: 10.1083/jcb.121.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Davis J., Bennett V. Brain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits. J Biol Chem. 1983 Jun 25;258(12):7757–7766. [PubMed] [Google Scholar]
  17. Davis L. H., Bennett V. Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin. J Biol Chem. 1990 Jun 25;265(18):10589–10596. [PubMed] [Google Scholar]
  18. Davis L. H., Otto E., Bennett V. Specific 33-residue repeat(s) of erythrocyte ankyrin associate with the anion exchanger. J Biol Chem. 1991 Jun 15;266(17):11163–11169. [PubMed] [Google Scholar]
  19. Díaz-Nido J., Serrano L., Méndez E., Avila J. A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation. J Cell Biol. 1988 Jun;106(6):2057–2065. doi: 10.1083/jcb.106.6.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fellous A., Francon J., Lennon A. M., Nunez J. Microtubule assembly in vitro. Purification of assembly-promoting factors. Eur J Biochem. 1977 Aug 15;78(1):167–174. doi: 10.1111/j.1432-1033.1977.tb11726.x. [DOI] [PubMed] [Google Scholar]
  21. Fischer G., Künemund V., Schachner M. Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surface glycoprotein L1. J Neurosci. 1986 Feb;6(2):605–612. doi: 10.1523/JNEUROSCI.06-02-00605.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goslin K., Schreyer D. J., Skene J. H., Banker G. Changes in the distribution of GAP-43 during the development of neuronal polarity. J Neurosci. 1990 Feb;10(2):588–602. doi: 10.1523/JNEUROSCI.10-02-00588.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Goslin K., Schreyer D. J., Skene J. H., Banker G. Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones. Nature. 1988 Dec 15;336(6200):672–674. doi: 10.1038/336672a0. [DOI] [PubMed] [Google Scholar]
  24. Hall T. G., Bennett V. Regulatory domains of erythrocyte ankyrin. J Biol Chem. 1987 Aug 5;262(22):10537–10545. [PubMed] [Google Scholar]
  25. Hisanaga S., Hirokawa N. Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing. J Mol Biol. 1988 Jul 20;202(2):297–305. doi: 10.1016/0022-2836(88)90459-7. [DOI] [PubMed] [Google Scholar]
  26. Hoshi M., Nishida E., Inagaki M., Gotoh Y., Sakai H. Activation of a serine/threonine kinase that phosphorylates microtubule-associated protein 1B in vitro by growth factors and phorbol esters in quiescent rat fibroblastic cells. Eur J Biochem. 1990 Oct 24;193(2):513–519. doi: 10.1111/j.1432-1033.1990.tb19366.x. [DOI] [PubMed] [Google Scholar]
  27. Inoue Y., Nakamura R., Mikoshiba K., Tsukada Y. Fine structure of the central myelin sheath in the myelin deficient mutant Shiverer mouse, with special reference to the pattern of myelin formation by oligodendroglia. Brain Res. 1981 Aug 24;219(1):85–94. doi: 10.1016/0006-8993(81)90269-9. [DOI] [PubMed] [Google Scholar]
  28. Julien J. P., Mushynski W. E. Multiple phosphorylation sites in mammalian neurofilament polypeptides. J Biol Chem. 1982 Sep 10;257(17):10467–10470. [PubMed] [Google Scholar]
  29. Julien J. P., Mushynski W. E. The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments. J Biol Chem. 1983 Mar 25;258(6):4019–4025. [PubMed] [Google Scholar]
  30. Kimura M., Inoko H., Katsuki M., Ando A., Sato T., Hirose T., Takashima H., Inayama S., Okano H., Takamatsu K. Molecular genetic analysis of myelin-deficient mice: shiverer mutant mice show deletion in gene(s) coding for myelin basic protein. J Neurochem. 1985 Mar;44(3):692–696. doi: 10.1111/j.1471-4159.1985.tb12870.x. [DOI] [PubMed] [Google Scholar]
  31. Kordeli E., Bennett V. Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice. J Cell Biol. 1991 Sep;114(6):1243–1259. doi: 10.1083/jcb.114.6.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kordeli E., Davis J., Trapp B., Bennett V. An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves. J Cell Biol. 1990 Apr;110(4):1341–1352. doi: 10.1083/jcb.110.4.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kunimoto M., Otto E., Bennett V. A new 440-kD isoform is the major ankyrin in neonatal rat brain. J Cell Biol. 1991 Dec;115(5):1319–1331. doi: 10.1083/jcb.115.5.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  35. Meiri K. F., Pfenninger K. H., Willard M. B. Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones. Proc Natl Acad Sci U S A. 1986 May;83(10):3537–3541. doi: 10.1073/pnas.83.10.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mikoshiba K., Kohsaka S., Takamatsu K., Tsukada Y. 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. 1981 Jan 12;204(2):455–460. doi: 10.1016/0006-8993(81)90608-9. [DOI] [PubMed] [Google Scholar]
  37. Noble M., Lewis S. A., Cowan N. J. The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau. J Cell Biol. 1989 Dec;109(6 Pt 2):3367–3376. doi: 10.1083/jcb.109.6.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Otto E., Kunimoto M., McLaughlin T., Bennett V. Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes. J Cell Biol. 1991 Jul;114(2):241–253. doi: 10.1083/jcb.114.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pachter J. S., Liem R. K. The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve. Dev Biol. 1984 May;103(1):200–210. doi: 10.1016/0012-1606(84)90021-6. [DOI] [PubMed] [Google Scholar]
  40. Rosenbluth J. Central myelin in the mouse mutant shiverer. J Comp Neurol. 1980 Dec 1;194(3):639–648. doi: 10.1002/cne.901940310. [DOI] [PubMed] [Google Scholar]
  41. Sato-Yoshitake R., Shiomura Y., Miyasaka H., Hirokawa N. Microtubule-associated protein 1B: molecular structure, localization, and phosphorylation-dependent expression in developing neurons. Neuron. 1989 Aug;3(2):229–238. doi: 10.1016/0896-6273(89)90036-6. [DOI] [PubMed] [Google Scholar]
  42. Skoff R. P., Price D. L., Stocks A. Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation. J Comp Neurol. 1976 Oct 1;169(3):291–312. doi: 10.1002/cne.901690303. [DOI] [PubMed] [Google Scholar]
  43. Skoff R. P., Price D. L., Stocks A. Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. II. Time of origin. J Comp Neurol. 1976 Oct 1;169(3):313–334. doi: 10.1002/cne.901690304. [DOI] [PubMed] [Google Scholar]
  44. Srinivasan Y., Lewallen M., Angelides K. J. Mapping the binding site on ankyrin for the voltage-dependent sodium channel from brain. J Biol Chem. 1992 Apr 15;267(11):7483–7489. [PubMed] [Google Scholar]
  45. Sternberger L. A., Sternberger N. H. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci U S A. 1983 Oct;80(19):6126–6130. doi: 10.1073/pnas.80.19.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Voter W. A., Erickson H. P. Electron microscopy of MAP 2 (microtubule-associated protein 2). J Ultrastruct Res. 1982 Sep;80(3):374–382. doi: 10.1016/s0022-5320(82)80051-8. [DOI] [PubMed] [Google Scholar]
  47. Vulliet P. R., Hall F. L., Mitchell J. P., Hardie D. G. Identification of a novel proline-directed serine/threonine protein kinase in rat pheochromocytoma. J Biol Chem. 1989 Sep 25;264(27):16292–16298. [PubMed] [Google Scholar]
  48. Wang D., Lewis S. A., Cowan N. J. Complete sequence of a cDNA encoding mouse MAP2. Nucleic Acids Res. 1988 Dec 9;16(23):11369–11370. doi: 10.1093/nar/16.23.11369. [DOI] [PMC free article] [PubMed] [Google Scholar]

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