Glycosylation of mammalian neurofilaments: Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M

Dennis L Y Dong, Zuo Shang Xu, Marc R. Chevrier, Robert J. Cotter, Don W. Cleveland, Gerald Warren Hart

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)16679-16687
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number22
StatePublished - Aug 5 1993

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Glycosylation
Acetylglucosamine
Intermediate Filaments
Proteolysis
Neurofilament Proteins
Peptides
Glycopeptides
Monosaccharides
Threonine
Stoichiometry
Serine
Purification
Mass spectrometry
Desorption
Gases
Degradation
Lasers
Head
Proteins
Axons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Glycosylation of mammalian neurofilaments : Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M. / Dong, Dennis L Y; Xu, Zuo Shang; Chevrier, Marc R.; Cotter, Robert J.; Cleveland, Don W.; Hart, Gerald Warren.

In: Journal of Biological Chemistry, Vol. 268, No. 22, 05.08.1993, p. 16679-16687.

Research output: Contribution to journalArticle

Dong, Dennis L Y ; Xu, Zuo Shang ; Chevrier, Marc R. ; Cotter, Robert J. ; Cleveland, Don W. ; Hart, Gerald Warren. / Glycosylation of mammalian neurofilaments : Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 22. pp. 16679-16687.
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T2 - Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M

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