Characterization of Recombinant and Brain Neuropsin, a Plasticity-related Serine Protease

Abstract

Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/ insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys 32 -Ile 33 bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.Some proteases have been suggested to be related to neural cell dynamics in such processes as cell death, migration, cellto-cell adhesion and de-adhesion, process elongation, pathfinding, and axonal rearrangement (1-5). These phenomena have been investigated by supplying known proteases involved in blood coagulation, fibrinolysis, or digestion to neural cell cultures. However, the observations that the proteases are mainly localized in and released from non-neural cells do not support all of such neural effects (5-7). Thus, we postulated that neurons themselves may produce and release their own proteases to participate in the neural cell dynamics described above. Neuropsin (NP)1 was cloned from the mouse brain and was shown to be localized in mouse hippocampal pyramidal neurons (8). These results and the observation that its mRNA showed marked activity-dependent changes caused by plasticity-inducible stimuli are suggestive of some neural effects in limbic plasticity (8, 9). However, it is still not known whether NP protein has enzyme activity as suggested by the deduced amino acid sequence (8). We postulated that the enzyme activity might be a molecular basis for the physiological responses induced by various stimuli. Therefore, in the present study, we examined whether recombinant NP (r-NP) and brain NP had proteolytic activity against synthetic and natural substrates. EXPERIMENTAL PROCEDURESMaterials-Mono S, Sepharose 2B, CNBr-activated Sepharose 4B and CL-6B, Superdex-75HR, Superose 12, Resource S, and Protein G-Sepharose were from Amersham Pharmacia Biotech. Silver staining kits were from Bio-Rad. Diisopropyl fluorophosphate (DFP), benzamidine, bestatin, soybean trypsin inhibitor, human plasma thrombin (EC 3.4.4.13), and TNM-FH insect cel...

Activation and enzymatic characterization of recombinant human kallikrein 8

“…Weak effects against trypsin-like activity have been reported for chymostatin, but these effects were always weaker than those observed for trypsin-like inhibitors (Jeohn et al, 1995;Valaitis, 1995). Chymostatin was also reported to have inhibitory activity against mK8, but the inhibitory activity was lower than for leupeptin (Shimizu et al, 1998). These data show hK8 inhibitor profile differences not only from trypsin, but also from mK8.…”

“…This difference is in agreement with the notion that hK8 prefers Arg at P1 position over Lys, and this preference might be stronger than that of mK8. Whereas the expression of mK8 in mouse brain was detected at the protein level (Shimizu et al, 1998), hK8 was not found to be expressed in appreciable amounts in brain tissue (Kishi et al, 2003b). These characteristic differences between hK8 and mK8 may indicate a difference in their physiological functions.…”

“…Indeed, recombinant hK7 and the mouse ortholog of hK8 (mK8) were previously shown to be activated by Lys-C (Hansson et al, 1994;Shimizu et al, 1998). Furthermore, crystallographic studies of the kallikreins hK1, hK6 and mK8 revealed very similar structures (Kishi et al, 1999;Bernett et al, 2002;Gomis-Ruth et al, 2002;Laxmikanthan et al, 2005).…”

“…Minor differences could be due to the different hK8 production protocol, since Rajapakse et al expressed the protease in E. coli and used a subsequent refolding protocol to obtain active hK8. mK8 was reported to show an up to five-fold preference for P1 Arg over Lys (Shimizu et al, 1998). …”

“…Therefore, the enzyme is believed to be associated with brain function, such as memory formation. Recombinant neuropsin was previously expressed in a baculovirus expression system and characterized as a trypsin-like protease, both enzymatically and structurally (Shimizu et al, 1998;Kishi et al, 1999). The human and rat orthologs were also cloned (Davies et al, 1998;Yoshida et al, 1998).…”