The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state.
Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150). Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights. Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion. Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200. A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase. Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well. The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase. The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged. We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007381:Intermediate Filament Proteins / Q000276:immunology / Q000378:metabolism*; D008854:Microscopy, Electron; D008970:Molecular Weight; D016900:Neurofilament Proteins; D010766:Phosphorylation; D013116:Spinal Cord / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D000469:Alkaline Phosphatase; D002918:Chymotrypsin