Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.

Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain

The degradation of phosphorylated and dephosphorylated neurofilament proteins by the Ca2+-activated neutral proteinase calpain was studied. Neurofilaments were isolated from bovine spinal cord, dephosphorylated by alkaline phosphatase (from Escherichia coli) and radioiodinated with [125I]-Bolton-Hunter reagent. The radioiodinated neurofilament proteins (untreated and dephosphorylated) were incubated in the presence and absence of calpain from rabbit skeletal muscle, and the degradation rates of large (NF-H), mid-sized (NF-M) and small (NF-L) neurofilament polypeptides were analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. The degradation of dephosphorylated neurofilament proteins occurred at a higher rate, and to a greater extent, than did that of the phosphorylated (untreated) neurofilament proteins. The dephosphorylated high-molecular-mass neurofilament (NF-HD) was proteolyzed 6 times more quickly than the untreated NF-H. The degradation rate of the NF-M and NF-L neurofilament proteins was also enhanced after dephosphorylation, but less than that of NF-H. This indicates that the dephosphorylation of neurofilament proteins can increase their sensitivity to calpain degradation.

Chemical cross-linking analyses of ox neurofilaments

Freshly isolated intact ox neurofilaments have been incubated with copper(II)-o-phenanthroline complex to induce thiol cross-linking between the two largest (apparent Mr 205 000 and 158 000) polypeptide components. Subsequent tryptic digestion shows that the thiol bonds formed between these polypeptides are distributed exclusively among ‘rod-domain’ fragments that remain associated with intact sedimentable filaments. These observations suggest that the polypeptide chains of the two largest neurofilament components are closely arranged within the backbone but are separate from one another in more peripheral regions. Soluble protofilaments derived from neurofilament disassembly at low ionic strength and high pH have also been cross-linked via thiol bonds in order to determine the polypeptide arrangement within these structures. All three neurofilament polypeptides cross-link more readily when in the form of protofilaments than when in the form of fully assembled filaments, and the pattern of cross-linked complexes formed is different. Analysis of one of these complexes shows that at least some of the protofilaments are composed of oligomers containing both the 72 000- and the 158 000-Mr neurofilament polypeptides arranged in close proximity.