An increasing number of studies indicate that
serine proteases play an important role in structural plasticity associated with learning and memory formation.
Neurotrypsin is a multidomain
serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions. A deletion in the
neurotrypsin gene causes severe
mental retardation in humans. For a biochemical characterization, we produced murine
neurotrypsin recombinantly in a eukaryotic expression system using myeloma cells. From the culture medium we purified
neurotrypsin using
heparin-, hydrophobic interaction- and immobilized
metal affinity chromatography. For an enzymological characterization two fragments of
agrin containing the natural cleavages sites of
neurotrypsin were used as substrates. The highest catalytic activity of
neurotrypsin was observed in the pH range between 7.0 and 8.5.
Calcium ions were required for
neurotrypsin activity and an ionic strength exceeding 500 mM decreased substrate cleavage. Site-specific mutations of the
amino acids flanking the scissile bonds showed that cleavage is highly specific and requires a
basic amino acid preceded by a
glutamate residue on the N-terminal side of the scissile bond. This sequence requirement argues for a unique substrate binding pocket of
neurotrypsin. This observation was further substantiated by the fact that almost all tested
serine protease inhibitors except dichloroisocoumarin and PMSF did not affect
neurotrypsin activity.