Serine Proteinase Inhibitor 3 and Murinoglobulin I Are Potent Inhibitors of Neuropsin in Adult Mouse Brain

Abstract

Extracellular serine protease neuropsin (NP) is expressed in the forebrain limbic area of adult brain and is implicated in synaptic plasticity. We screened for endogenous NP inhibitors with recombinant NP (r-NP) from extracts of the hippocampus and the cerebral cortex in adult mouse brain. Two SDS-stable complexes were detected, and after their purification, peptide sequences were determined by amino acid sequencing and mass spectrometry, revealing that target molecules were serine proteinase inhibitor-3 (SPI3) and murinoglobulin I (MUG I). The addition of the recombinant SPI3 to r-NP resulted in an SDS-stable complex, and the complex formation followed bimolecular kinetics with an association rate constant of 3.4 ؎ 0.22 ؋ 10 6 M ؊1 s ؊1, showing that SPI3 was a slow, tight binding inhibitor of NP. In situ hybridization histochemistry showed that SPI3 mRNA was expressed in pyramidal neurons in the hippocampal CA1-CA3 subfields, as was NP mRNA. Alternatively, the addition of purified plasma MUG I to r-NP resulted in an SDS-stable complex, and MUG I inhibited degradation of fibronectin by r-NP to 24% at a r-NP/MUG I molar ratio of 1:2. Immunofluorescence histochemistry showed that MUG I localized in the hippocampal neurons. These findings indicate that SPI3 and MUG I serve to inactivate NP and control the level of NP in adult brain, respectively. Extracellular proteolysis exerted by secretory serine proteases has been implicated in neural development, plasticity, and degeneration and regeneration in the nervous system (1) and might be controlled by specific inhibitors (2). Neuropsin (NP), 1 a serine protease with a chimeric structure similar to trypsin and nerve growth factor-␥ (3), was found to be expressed in the nervous system (4) and has been demonstrated to be engaged in activity-dependent plasticity changes in neurons. NP mRNA and protein levels increased in the hippocampus after kindled seizures and injection of antibody against NP led to retardation of epilepticus in mice (5, 6). Furthermore, application of recombinant NP induced an increase in the amplitude of the tetanic stimulation-induced early phase long term potentiation in the Schaffer collateral pathway (7). It has been proposed that the plasticity changes are regulated by the balance between the accumulation and degradation of the extracellular matrix (ECM) proteins. There is, indeed, some evidence that the formation of hippocampal LTP is attributable to cell-ECM interactions, involving cadherin (8), integrin (9, 10), N-syndecan (11), cell adhesion molecules, NCAM, and L1 (12)(13)(14). NP acted to degrade ECM including fibronectin (15), for which integrins were receptors (16, 17), and L1. 2 Therefore, rearrangement of these ECM components by NP and a specific inhibitor might implicated in the formation of LTP.As another characteristic of NP, it has been shown that NP mRNA was restricted to neurons in the limbic areas of adult brain involving the CA1-CA3 subfields of the hippocampus, the amygdaloid nucleus, the cingulate cortex, the anterior olfact...

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