Crystal Structure of Neuropsin, a Hippocampal Protease Involved in Kindling Epileptogenesis

Abstract

Neuropsin is a novel serine protease, the expression of which is highly localized in the limbic areas of the mouse brain and which is suggested to be involved in kindling epileptogenesis and hippocampal plasticity. The 2.1-Å resolution crystal structure of neuropsin provides the first three-dimensional view of one of the serine proteases highly expressed in the nervous system, and reveals a serine protease fold that exhibits chimeric features between trypsin and nerve growth factor-␥ (NGF␥), a member of the kallikrein family. Neuropsin possesses an N-glycosylated "kallikrein loop" but forms six disulfide bonds corresponding to those of trypsin. The ordered kallikrein loop projects proline toward the active site to restrict smaller residues or proline at the P2 position of substrates. Loop F, which participates in forming the S3/S4 sites, is similar to trypsin rather than NGF␥. The unique conformations of loops G and H form an S1 pocket specific for both arginine and lysine. These characteristic loop structures forming the substratebinding site suggest the novel substrate specificity of neuropsin and give a clue to the design of its specific inhibitors.Proteases have been shown to play essential roles in the nervous system, including those of neurite outgrowth (1), neural degeneration (2), and synaptic plasticity (3). These actions are thought to be mediated by the proteolytic cleavage of zymogen precursors, the activation of specific cell surface receptors, or the degradation of extracellular matrix proteins (4). Neuropsin was cloned from a mouse hippocampal cDNA library using sequences for key regions of the serine protease domain of nerve growth factor (NGF)-␥ 1 (5). In the brain, no NGF␥ has been identified so far, though NGF␤ is present. Neuropsin is one of the serine proteases highly expressed in the nervous system (6 -8). The expression of neuropsin is localized at highest concentration in the hippocampus and the amygdala, which are important for acquisition of memory and emotional memory, respectively. This localization is in contrast to that of tissue plasminogen activator (tPA), which is well documented to play a crucial role in the nervous system by mediating plasticity but is distributed more uniformly across the other brain regions and throughout other organs (9). Activity-dependent changes in expression of neuropsin have been observed upon direct hippocampal stimulation and induction of kindling, which is a model for epilepsy and neuronal plasticity characterized by the progressive development of electrographic and behavioral seizures (5, 10). A single intraventricular injection of monoclonal antibodies specific to neuropsin reduces or eliminates the epileptic pattern (11). Moreover, oxidative stress is shown to effect the expression of neuropsin in the limbic areas, which might be related to the disturbance in shockavoidance learning of mice (12). These activity-dependent changes and the specific localization of neuropsin indicate the involvement of this protease in hippocampal plasticity and its p...

Activation and enzymatic characterization of recombinant human kallikrein 8

“…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). From sequence analysis of other kallikreins, it is believed that all of them share a very close ternary structure.…”

“…As described for these proteases, increased hK8 activity may be caused by stabilization of the enzyme structure induced by Ca 2q binding. Amino-acid sequence alignment of trypsin, mK8 and hK8, and structural comparison of trypsin and mK8 Kishi et al, 1999) showed no insertions or deletions within the Ca 2q -binding loop region among these enzymes. Using structural analysis of trypsin complexed with Ca 2q (Bode and Schwager, 1975), it was shown that this ion associates with five amino acids on a loop of trypsin with five coordinate bonds.…”

“…Active hK8 contains a total of 15 Lys residues, three of which are positioned close to the active site of the enzyme . In the mouse ortholog, these Lys residues are predicted to be on a loop region (Kishi et al, 1999). Cleavage within this region would give rise to a C-terminal fragment of approximately 25 kDa, corresponding in size to the band detected by anti-hK8 antibody after prolonged incubation of hK8 with Lys-C ( Figure 1B).…”

“…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).…”

The Structure of Human Prokallikrein 6 Reveals a Novel Activation Mechanism for the Kallikrein Family

“…This protein may have orthologues in mouse, rat, and monkey (28 -31). It is distinct from neuropsin (KLK8), a hippocampal protease involved in kindling epileptogenesis in mice (32). Like the other members of the human kallikrein family, the KLK6 gene lies at chromosome locus 19q13.4, telomeric to the PSA (KLK3) and KLK2 genes (5,21).…”