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.
Highlights
From the Departments of $Biobgica/ Chemistry, llPharmacology and Molecular Sciences, and **Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
NF Polypeptides Are Glycosylated by 0-Linked GkNAcTotal NF proteins comprising primarily the three NF subunits, NF-L, NF-M, and NF-H, were partially purified from rat spinal cord (Fig. 1,lane 1).NF-L and NF-M were purified to homogeneity (Fig. 1(left),lanes 2 and 3)
To see whether NF polypeptides are glycosylated, total NFs (25 pg), purified NF-M (5 pg), and NF-L (5 pg) protein were separately labeled with UDP-[3H]galactose and galactosyltransferase, analyzed by SDS-PAGE, and fluorographed
Summary
Peptides-Total NFs, purified NF-M or NF-L, or trypticNF peptides (see below) waslabeled with UDP-['H]galactose and galactosyltransferase as described [20] with the following changes. Manual Edman degradation sequencing of glycopeptides was performed by covalent coupling of peptides to Sequelon-AA membranes (MilliGen/Biosearch of Millipore), followedby repeatedly coupling and trifluoroacetic acid extraction as described [41] except that theproduct from each cycle was dried and neutralized before scintillation counting. A trace amount of [3H]glucosamine(50,000cpm, 38.5 Ci/mmol) was added to each sample prior to hydrolysis as an internal standardfor measuring recovery and for followingthe amino sugars durincgolumn separation (this amount of tracer is too little amountto be detected by Dionex HPAEC-PAD analysis). After hydrolysis, those samples were dried under avortex-evaporator, resuspended in 0.5 mlof water. 8.0% SDS-PAGE [43], stainewd ith CoomassieBrilliant Blue, treated with En3Hance (Du Pont-New England Nuclear), dried, and fluorographed
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