O-glycosylation of the tail domain of neurofilament protein M in human neurons and in spinal cord tissue of a rat model of amyotrophic lateral sclerosis (ALS)

J Biol Chem. 2005 Sep 9;280(36):31648-58. doi: 10.1074/jbc.M504395200. Epub 2005 Jul 8.

Abstract

Mammalian neurofilaments (NFs) are modified by post-translational modifications that are thought to regulate NF assembly and organization. Whereas phosphorylation has been intensely studied, the role of another common modification, the attachment of O-linked N-acetylglucosamine (GlcNAc) to individual serine and threonine residues, is hardly understood. We generated a novel monoclonal antibody that specifically recognizes an O-glycosylated epitope in the tail domain of NF-M and allows determination of the glycosylation state at this residue. The antibody displays strong species preference for human NF-M, shows some reactivity with rat but not with mouse or bovine NF-M. By immunohistochemistry and Western blot analysis of biopsy-derived human temporal lobe tissue we show that immunoreactivity is highly enriched in axons parallel to hyperphosphorylated NFs. Treatment of cultured neurons with the GlcNAcase inhibitor PUGNAc causes a 40% increase in immunoreactivity within 1 h, which is completely reversible and parallels the total increase in cellular O-GlcNAc modification. Treatment with the mitogen-activated protein kinase kinase inhibitor PD-98059 leads to a similar increase in immunoreactivity. In spinal cord tissue of a transgenic rat model for amyotrophic lateral sclerosis, immunoreactivity is strongly decreased compared with wild-type animals while phosphorylation is increased. The data suggest that hyperphosphorylation and tail domain O-glycosylation of NFs are synchronously regulated in axons of human neurons in situ and that O-glycosylation of NF-M is highly dynamic and closely interweaved with phosphorylation cascades and may have a pathophysiological role.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosaminidase / antagonists & inhibitors
  • Amino Acid Sequence
  • Amyotrophic Lateral Sclerosis / metabolism*
  • Animals
  • Animals, Genetically Modified
  • Antibodies, Monoclonal
  • Axons / metabolism
  • Cattle
  • Cell Line, Tumor
  • Disease Models, Animal
  • Epitopes / immunology
  • Glycosylation
  • Humans
  • Mice
  • Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors
  • Molecular Sequence Data
  • NIH 3T3 Cells
  • Neurofilament Proteins / immunology
  • Neurofilament Proteins / metabolism*
  • Neurons / metabolism*
  • Protein Structure, Tertiary
  • Rats
  • Spinal Cord / metabolism*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • Neurofilament Proteins
  • neurofilament protein M
  • Mitogen-Activated Protein Kinase Kinases
  • Acetylglucosaminidase