Degradation of Neurofilament Proteins by Purified Human Brain Cathepsin D

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Abstract
Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFP) prepared by axonal flotation led to the rapid degradation of the 200K [kilodalton], 160K and 70K NFP subunits which were separated by 1- or 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was appreciable at enzyme activity-to-substrate protein ratios that were 2- to 3-fold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFP separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP breakdown products, antisera against each of the major NFP were applied to nitrocellulose electroblots of NFP separated by 2-dimensional SDS-PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno-cross-reactive fragments generated from NFP by CD comigrated on 2-dimensional gels with polypeptides present in unincubated preparations. NFP and other cytoskeletal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed.