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. 1987 Jun;6(6):1617–1626. doi: 10.1002/j.1460-2075.1987.tb02409.x

The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family.

M W Myers, R A Lazzarini, V M Lee, W W Schlaepfer, D L Nelson
PMCID: PMC553533  PMID: 3608989

Abstract

We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF-M. Analysis of the sequence has allowed us to investigate the structure of the carboxy-terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxy-terminal region of the protein contains a 13 amino acid proline- and serine-rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys-Ser-Pro-Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled-coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurofilament genes of mammals have similar structures which are quite different from those of the other intermediate filaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.

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  1. Allison L. A., Moyle M., Shales M., Ingles C. J. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell. 1985 Sep;42(2):599–610. doi: 10.1016/0092-8674(85)90117-5. [DOI] [PubMed] [Google Scholar]
  2. Angeletti R. H., Trojanowski J. Q., Carden M., Schlaepfer W. W., Lee V. M. Domain structure of neurofilament subunits as revealed by monoclonal antibodies. J Cell Biochem. 1985;27(2):181–187. doi: 10.1002/jcb.240270212. [DOI] [PubMed] [Google Scholar]
  3. Balcarek J. M., Cowan N. J. Structure of the mouse glial fibrillary acidic protein gene: implications for the evolution of the intermediate filament multigene family. Nucleic Acids Res. 1985 Aug 12;13(15):5527–5543. doi: 10.1093/nar/13.15.5527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett G. S., DiLullo C. Slow posttranslational modification of a neurofilament protein. J Cell Biol. 1985 May;100(5):1799–1804. doi: 10.1083/jcb.100.5.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carden M. J., Schlaepfer W. W., Lee V. M. The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state. J Biol Chem. 1985 Aug 15;260(17):9805–9817. [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. Craig R., Smith R., Kendrick-Jones J. Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules. 1983 Mar 31-Apr 6Nature. 302(5907):436–439. doi: 10.1038/302436a0. [DOI] [PubMed] [Google Scholar]
  8. Dawson P. J., Hulme J. S., Lloyd C. W. Monoclonal antibody to intermediate filament antigen cross-reacts with higher plant cells. J Cell Biol. 1985 May;100(5):1793–1798. doi: 10.1083/jcb.100.5.1793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fisher D. Z., Chaudhary N., Blobel G. cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6450–6454. doi: 10.1073/pnas.83.17.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  11. Geisler N., Fischer S., Vandekerckhove J., Damme J. V., Plessmann U., Weber K. Protein-chemical characterization of NF-H, the largest mammalian neurofilament component; intermediate filament-type sequences followed by a unique carboxy-terminal extension. EMBO J. 1985 Jan;4(1):57–63. doi: 10.1002/j.1460-2075.1985.tb02317.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geisler N., Fischer S., Vandekerckhove J., Plessmann U., Weber K. Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension. EMBO J. 1984 Nov;3(11):2701–2706. doi: 10.1002/j.1460-2075.1984.tb02196.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geisler N., Kaufmann E., Fischer S., Plessmann U., Weber K. Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins. EMBO J. 1983;2(8):1295–1302. doi: 10.1002/j.1460-2075.1983.tb01584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geisler N., Plessmann U., Weber K. The complete amino acid sequence of the major mammalian neurofilament protein (NF-L). FEBS Lett. 1985 Mar 25;182(2):475–478. doi: 10.1016/0014-5793(85)80357-4. [DOI] [PubMed] [Google Scholar]
  15. Geisler N., Weber K. Comparison of the proteins of two immunologically distinct intermediate-sized filaments by amino acid sequence analysis: desmin and vimentin. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4120–4123. doi: 10.1073/pnas.78.7.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geisler N., Weber K. Self-assembly in Vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments. J Mol Biol. 1981 Sep 25;151(3):565–571. doi: 10.1016/0022-2836(81)90011-5. [DOI] [PubMed] [Google Scholar]
  17. Glenner G. G., Wong C. W. Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 1984 May 16;120(3):885–890. doi: 10.1016/s0006-291x(84)80190-4. [DOI] [PubMed] [Google Scholar]
  18. Hanukoglu I., Fuchs E. The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins. Cell. 1982 Nov;31(1):243–252. doi: 10.1016/0092-8674(82)90424-x. [DOI] [PubMed] [Google Scholar]
  19. Hirokawa N., Glicksman M. A., Willard M. B. Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J Cell Biol. 1984 Apr;98(4):1523–1536. doi: 10.1083/jcb.98.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hood L., Steinmetz M., Malissen B. Genes of the major histocompatibility complex of the mouse. Annu Rev Immunol. 1983;1:529–568. doi: 10.1146/annurev.iy.01.040183.002525. [DOI] [PubMed] [Google Scholar]
  21. Huxley H. E. The mechanism of muscular contraction. Science. 1969 Jun 20;164(3886):1356–1365. doi: 10.1126/science.164.3886.1356. [DOI] [PubMed] [Google Scholar]
  22. Johnson L. D., Idler W. W., Zhou X. M., Roop D. R., Steinert P. M. Structure of a gene for the human epidermal 67-kDa keratin. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1896–1900. doi: 10.1073/pnas.82.7.1896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Julien J. P., Meyer D., Flavell D., Hurst J., Grosveld F. Cloning and developmental expression of the murine neurofilament gene family. Brain Res. 1986 Dec;387(3):243–250. doi: 10.1016/0169-328x(86)90030-6. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Julien J. P., Ramachandran K., Grosveld F. Cloning of a cDNA encoding the smallest neurofilament protein from the rat. Biochim Biophys Acta. 1985 Aug 21;825(4):398–404. doi: 10.1016/0167-4781(85)90067-3. [DOI] [PubMed] [Google Scholar]
  27. Julien J. P., Smoluk G. D., Mushynski W. E. Characteristics of the protein kinase activity associated with rat neurofilament preparations. Biochim Biophys Acta. 1983 Jan 4;755(1):25–31. doi: 10.1016/0304-4165(83)90268-4. [DOI] [PubMed] [Google Scholar]
  28. Kamholz J., de Ferra F., Puckett C., Lazzarini R. Identification of three forms of human myelin basic protein by cDNA cloning. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4962–4966. doi: 10.1073/pnas.83.13.4962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Karin M., Richards R. I. Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene. Nature. 1982 Oct 28;299(5886):797–802. doi: 10.1038/299797a0. [DOI] [PubMed] [Google Scholar]
  30. Kaufmann E., Geisler N., Weber K. SDS-PAGE strongly overestimates the molecular masses of the neurofilament proteins. FEBS Lett. 1984 May 7;170(1):81–84. doi: 10.1016/0014-5793(84)81373-3. [DOI] [PubMed] [Google Scholar]
  31. Lasek R. J., Phillips L., Katz M. J., Autilio-Gambetti L. Function and evolution of neurofilament proteins. Ann N Y Acad Sci. 1985;455:462–478. doi: 10.1111/j.1749-6632.1985.tb50429.x. [DOI] [PubMed] [Google Scholar]
  32. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature. 1980 Jan 17;283(5744):249–256. doi: 10.1038/283249a0. [DOI] [PubMed] [Google Scholar]
  33. Lee V. M., Carden M. J., Schlaepfer W. W. Structural similarities and differences between neurofilament proteins from five different species as revealed using monoclonal antibodies. J Neurosci. 1986 Aug;6(8):2179–2186. doi: 10.1523/JNEUROSCI.06-08-02179.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lee V. M., Carden M. J., Trojanowski J. Q. Novel monoclonal antibodies provide evidence for the in situ existence of a nonphosphorylated form of the largest neurofilament subunit. J Neurosci. 1986 Mar;6(3):850–858. doi: 10.1523/JNEUROSCI.06-03-00850.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Leterrier J. F., Liem R. K., Shelanski M. L. Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging. J Cell Biol. 1982 Dec;95(3):982–986. doi: 10.1083/jcb.95.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lewis S. A., Cowan N. J. Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum. Mol Cell Biol. 1986 May;6(5):1529–1534. doi: 10.1128/mcb.6.5.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lewis S. A., Cowan N. J. Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe. J Cell Biol. 1985 Mar;100(3):843–850. doi: 10.1083/jcb.100.3.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Luca F. C., Bloom G. S., Vallee R. B. A monoclonal antibody that cross-reacts with phosphorylated epitopes on two microtubule-associated proteins and two neurofilament polypeptides. Proc Natl Acad Sci U S A. 1986 Feb;83(4):1006–1010. doi: 10.1073/pnas.83.4.1006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Marchuk D., McCrohon S., Fuchs E. Remarkable conservation of structure among intermediate filament genes. Cell. 1984 Dec;39(3 Pt 2):491–498. doi: 10.1016/0092-8674(84)90456-2. [DOI] [PubMed] [Google Scholar]
  40. Masters C. L., Simms G., Weinman N. A., Multhaup G., McDonald B. L., Beyreuther K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4245–4249. doi: 10.1073/pnas.82.12.4245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  42. McKeon F. D., Kirschner M. W., Caput D. Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature. 1986 Feb 6;319(6053):463–468. doi: 10.1038/319463a0. [DOI] [PubMed] [Google Scholar]
  43. Miake-Lye R., Kirschner M. W. Induction of early mitotic events in a cell-free system. Cell. 1985 May;41(1):165–175. doi: 10.1016/0092-8674(85)90071-6. [DOI] [PubMed] [Google Scholar]
  44. Miller C. C., Brion J. P., Calvert R., Chin T. K., Eagles P. A., Downes M. J., Flament-Durand J., Haugh M., Kahn J., Probst A. Alzheimer's paired helical filaments share epitopes with neurofilament side arms. EMBO J. 1986 Feb;5(2):269–276. doi: 10.1002/j.1460-2075.1986.tb04209.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Minami Y., Sakai H. Dephosphorylation suppresses the activity of neurofilament to promote tubulin polymerization. FEBS Lett. 1985 Jun 17;185(2):239–242. doi: 10.1016/0014-5793(85)80914-5. [DOI] [PubMed] [Google Scholar]
  46. Powell B. C., Cam G. R., Fietz M. J., Rogers G. E. Clustered arrangement of keratin intermediate filament genes. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5048–5052. doi: 10.1073/pnas.83.14.5048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Quax W., Egberts W. V., Hendriks W., Quax-Jeuken Y., Bloemendal H. The structure of the vimentin gene. Cell. 1983 Nov;35(1):215–223. doi: 10.1016/0092-8674(83)90224-6. [DOI] [PubMed] [Google Scholar]
  48. Quax W., van den Broek L., Egberts W. V., Ramaekers F., Bloemendal H. Characterization of the hamster desmin gene: expression and formation of desmin filaments in nonmuscle cells after gene transfer. Cell. 1985 Nov;43(1):327–338. doi: 10.1016/0092-8674(85)90038-8. [DOI] [PubMed] [Google Scholar]
  49. Rackwitz H. R., Zehetner G., Frischauf A. M., Lehrach H. Rapid restriction mapping of DNA cloned in lambda phage vectors. Gene. 1984 Oct;30(1-3):195–200. doi: 10.1016/0378-1119(84)90120-3. [DOI] [PubMed] [Google Scholar]
  50. RayChaudhury A., Marchuk D., Lindhurst M., Fuchs E. Three tightly linked genes encoding human type I keratins: conservation of sequence in the 5'-untranslated leader and 5'-upstream regions of coexpressed keratin genes. Mol Cell Biol. 1986 Feb;6(2):539–548. doi: 10.1128/mcb.6.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Robinson P. A., Wion D., Anderton B. H. Isolation of a cDNA for the rat heavy neurofilament polypeptide (NF-H). FEBS Lett. 1986 Dec 15;209(2):203–205. doi: 10.1016/0014-5793(86)81111-5. [DOI] [PubMed] [Google Scholar]
  52. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  53. Soares M. B., Schon E., Henderson A., Karathanasis S. K., Cate R., Zeitlin S., Chirgwin J., Efstratiadis A. RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon. Mol Cell Biol. 1985 Aug;5(8):2090–2103. doi: 10.1128/mcb.5.8.2090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Steinert P. M., Steven A. C., Roop D. R. The molecular biology of intermediate filaments. Cell. 1985 Sep;42(2):411–420. doi: 10.1016/0092-8674(85)90098-4. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Stone D., Smillie L. B. The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence. J Biol Chem. 1978 Feb 25;253(4):1137–1148. [PubMed] [Google Scholar]
  57. Tyner A. L., Eichman M. J., Fuchs E. The sequence of a type II keratin gene expressed in human skin: conservation of structure among all intermediate filament genes. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4683–4687. doi: 10.1073/pnas.82.14.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zimmerman U. J., Schlaepfer W. W. Clustering of phosphorylated amino acid residues in neurofilament proteins as revealed by 31P NMR. Biochemistry. 1986 Jun 17;25(12):3533–3536. doi: 10.1021/bi00360a009. [DOI] [PubMed] [Google Scholar]

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