Phosphorylation state of the native high‐molecular‐weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis

• Published in: Journal of neurochemistry Vol. 76; no. 5; pp. 1315 - 1325
• Main Authors: Strong, Michael J., Strong, Wendy L., Jaffe, Howard, Traggert, Bearnd, Sopper, Maggie M., Pant, Harish C.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-03-2001; Blackwell
• Subjects: Amino Acid Sequence; amyotrophic lateral sclerosis; Biological and medical sciences; Calmodulin - metabolism; Calpain - metabolism; Cervical Vertebrae; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Electrophoresis, Gel, Two-Dimensional; Humans; Isoelectric Focusing; Medical sciences; Molecular Sequence Data; motor neuron disease; Motor Neuron Disease - metabolism; neurofilament; Neurofilament Proteins - chemistry; Neurofilament Proteins - isolation & purification; Neurofilament Proteins - metabolism; Neurology; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Phosphopeptides - chemistry; Phosphopeptides - isolation & purification; Phosphoproteins - chemistry; Phosphorylation; Protein Subunits; Reference Values; Spinal Cord - chemistry; Human; Nervous system diseases; Spinal cord; Phosphorylation; Central nervous system; Motor neuron disease; Amyotrophic lateral sclerosis; Subunit; Genetic disease; Neurofilament; Central nervous system disease; Degenerative disease; Spinal cord disease
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2001.00094.x

The intraneuronal aggregation of phosphorylated high‐molecular‐weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper‐phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X‐100 insoluble protein fractions, with respect to migration patterns on 2D‐iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain‐mediated proteolysis. The rate of calpain‐mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.