title: Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments.
abstract: The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one-dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton-insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont, Massachusetts 02178.
authors.forename: S E
authors.initials: SE
authors.lastname: Lewis
authors.forename: R A
authors.initials: RA
authors.lastname: Nixon
mesh_terms: D000818:Animals; D001345:Autoradiography; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D016900:Neurofilament Proteins; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D010449:Peptide Mapping; D010766:Phosphorylation; D012160:Retina / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins