title: Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M.
abstract: Neurofilaments are neuronal intermediate filaments that play an important role in the growth and maintenance of large myelinated axons. Mammalian neurofilaments are composed of three polypeptide subunits, designed as NF-L, NF-M, and NF-H, all of which are phosphorylated. Here, we demonstrate by several criteria that neurofilament polypeptides are also modified by an abundant type of intracellular protein glycosylation in which single N-acetylglucosamine monosaccharides are O-glycosidically (O-GlcNAc) linked to serine or threonine residues. In purified neurofilament proteins, the O-GlcNAc modifications occur at a stoichiometry of approximately 0.1 and 0.15 mol of GlcNAc/mol of NF-L and NF-M, respectively. The predominant sites of O-GlcNAc attachment on NF-L and NF-M are identified using proteolysis, purification of the glycopeptides, and subsequent analysis by automated gas-phase sequencing, manual Edman degradation, and laser desorption mass spectrometry. For NF-L, both major sites of glycosylation (Thr21 and Ser27) are located at the NH2-terminal head domain. For NF-M, one major site (Thr48) lies within the NH2-terminal head domain, whereas the other (Thr431) is located at the tail domain. Deletions encompassing these sites have been shown previously to have a dominant detrimental effect upon neurofilament assembly, raising questions about the specific function(s) of the saccharide moieties at these sites. Specific identification of these O-GlcNAc attachment sites has set the stage for more detailed mutagenic analysis of O-GlcNAc functions on neurofilaments.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: D L
authors.initials: DL
authors.lastname: Dong
authors.forename: Z S
authors.initials: ZS
authors.lastname: Xu
authors.forename: M R
authors.initials: MR
authors.lastname: Chevrier
authors.forename: R J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: D W
authors.initials: DW
authors.lastname: Cleveland
authors.forename: G W
authors.initials: GW
authors.lastname: Hart
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: D016900:Neurofilament Proteins; C044882:neurofilament protein L; C061195:neurofilament protein M; D000117:Acetylglucosamine