Abstract
BackgroundTau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA). Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations.ResultsAn N-terminally His tagged-PSA was expressed and purified from Sf9 insect cells. Although this PSA preparation cleaved Tau, product analysis with N and C terminal Tau antibodies coupled with mass spectrometry showed an endoproteolytic cleavage atypical for an aminopeptidase. Furthermore, the reaction was not blocked by the general aminopeptidase inhibitor bestatin or the specific PSA inhibitor puromycin. In order to test whether Tau hydrolysis might be caused by a protease contaminant the enzyme was expressed in E. coli as glutathione S-transferase and maltose binding protein fusion proteins or in Sf9 cells as a C-terminally His-tagged protein. After purification to near homogeneity none of these other recombinant forms of PSA cleaved Tau. Further, Tau-cleaving activity and aminopeptidase activities derived from the Sf9 cell expression system were separable by molecular sieve chromatography. When tested in a cellular context we again failed to see a PSA dependent cleavage of Tau. A commercial preparation of a related aminopeptidase, aminopeptidase N, also exhibited Tau cleaving activity, but this activity could also be separated from aminopeptidase activity.ConclusionIt is concluded that PSA does not directly cleave Tau.
Highlights
The microtubule-associated protein tau (Tau) is located primarily in the central nervous system (CNS) and regulates the stability of microtubules
We initially tested the reaction of purified recombinant human puromycin sensitive aminopeptidase (PSA) expressed in Sf9 insect cells with Tau
Since hPSASf is an exopeptidase that cleaves amino acids sequentially from the N-terminus, we were surprised to find that reaction products, N1 to N3, which were decreased in size by apparent molecular weights of greater than 15 kDa, still retained the epitopes for the N-terminally directed antibody 5A6
Summary
We initially tested the reaction of purified recombinant human PSA expressed in Sf9 insect cells with Tau. Since N-His6-hPSA did not bind well to a nickel resin and was purified conventionally, an additional form of PSA containing a C-terminal His tag (C-His6PSA) was expressed in Sf9 insect cells and purified on a Figure 3 Hydrolysis of Tau by hPSASf in the presence of protease inhibitors. This shows the presence of endogenous Tau cleaving activity in Sf9 cells that likely contaminated our purified N-His6-hPSASf preparation. The results showed that aminopeptidase activity and Tau-cleaving activity are clearly resolved (figure 7), demonstrating that aminopeptidase N does not hydrolyze Tau. Attempts to identify the Tau-cleaving enzyme by mass spectral fingerprinting met the same difficulty as with the insect protein, namely a lack of an adequate database. The identification of the Tau cleaving enzymes derived both Sf9 cells and from porcine kidney remains unknown, but is clearly not PSA
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