title: A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF-M and NF-H.
abstract: We have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C-terminal "tail" sequences of the two high molecular weight neurofilament subunits, NF-M and NF-H. A series of recombinant fusion proteins containing selected NF-M and NF-H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS-PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF-M and NF-H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF-M and NF-H sequence. These antibody studies showed that the extreme C-termini of NF-M and NF-H are immunologically absolutely distinct from one another and also indicate that the extreme C-terminus of NF-M is immunologically much more conserved than the analogous region of NF-H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C-terminus of NF-M. We also show that the unusual immunological properties of the bovine NF-M tail we have previously observed do not extend to the extreme C-terminal region, which appears immunologically no different from the analogous region of other NF-M molecules. The peculiarities of bovine NF-M could be explained by the presence of a KSP motif that resembles the NF-H KSP prototype.
authors.affiliation: Department of Neuroscience, University of Florida College of Medicine, Gainesville 32610.
authors.forename: J
authors.initials: J
authors.lastname: Harris
authors.forename: C
authors.initials: C
authors.lastname: Ayyub
authors.forename: G
authors.initials: G
authors.lastname: Shaw
mesh_terms: D000818:Animals; D001369:Axons / Q000648:ultrastructure; D001483:Base Sequence; D001921:Brain / Q000166:cytology / Q000502:physiology; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011919:Rats, Inbred Strains; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000939:Epitopes; D046911:Macromolecular Substances; D016900:Neurofilament Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C051774:neurofilament protein H; C061195:neurofilament protein M; D004247:DNA