Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser55, Ser66, and Ser472) and NFM has 22 (Ser512, Ser546, Ser554, Ser560, Thr627, Ser629, Ser634, Ser639, Thr646, Ser649, Ser654, Ser664, Ser669, Thr676, Ser679, Ser684, Ser694, Ser726, Ser750, Ser756, Ser770, and Ser846) and two tentative sites (Ser659/Thr661 and Thr840). Ser66 was previously not known to be phosphorylated in NFL of other species, while two sites (Ser55 and Ser472) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser55, Ser66, Ser 472, are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser 512, Thr627, Ser629, Ser634, Ser639, Thr646, Ser649, Ser654, Ser664, Ser669, Thr676, Ser679, Ser684, Ser694, Ser726, and Ser750), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser546, Ser554, Ser560, Ser756, Ser770, and Ser846). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.
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