Zymogen activation of neurotrypsin and neurotrypsin-dependent agrin cleavage on the cell surface are enhanced by glycosaminoglycans.

The serine peptidase neurotrypsin is stored in presynaptic nerve endings and secreted in an inactive zymogenic form by synaptic activity. After activation, which requires activity of postsynaptic NMDA (N-methyl-D-aspartate) receptors, neurotrypsin cleaves the heparan sulfate proteoglycan agrin at active synapses. The resulting C-terminal 22-kDa fragment of agrin induces dendritic filopodia, which are considered to be precursors of new synapses. In the present study, we investigated the role of GAGs (glycosaminoglycans) in the activation of neurotrypsin and neurotrypsin-dependent agrin cleavage. We found binding of neurotrypsin to the GAG side chains of agrin, which in turn enhanced the activation of neurotrypsin by proprotein convertases and resulted in enhanced agrin cleavage. A similar enhancement of neurotrypsin binding to agrin, neurotrypsin activation and agrin cleavage was induced by the four-amino-acid insert at the y splice site of agrin, which is crucial for the formation of a heparin-binding site. Non-agrin GAGs also contributed to binding and activation of neurotrypsin and, thereby, to agrin cleavage, albeit to a lesser extent. Binding of neurotrypsin to cell-surface glycans locally restricts its conversion from zymogen into active peptidase. This provides the molecular foundation for the local action of neurotrypsin at or in the vicinity of its site of synaptic secretion. By its local action at synapses with correlated pre- and post-synaptic activity, the neurotrypsin-agrin system fulfils the requirements for a mechanism serving experience-dependent modification of activated synapses, which is essential for adaptive structural reorganizations of neuronal circuits in the developing and/or adult brain.

Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation.

The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin-deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites. Neurotrypsin and agrin were expressed recombinantly, purified, and assayed in vitro. A catalytic efficiency of 1.3 x 10(4) M(-1) x s(-1) was determined. Neurotrypsin activity was shown to depend on calcium with an optimal activity in the pH range of 7-8.5. Mutagenesis analysis of the amino acids flanking the scissile bonds showed that cleavage is highly specific due to the unique substrate recognition pocket of neurotrypsin at the active site. The C-terminal agrin fragment released after cleavage has recently been identified as an inactivating ligand of the Na+/K+-ATPase at CNS synapses, and its binding has been demonstrated to regulate presynaptic excitability. Therefore, dysregulation of agrin processing is a good candidate for a pathogenetic mechanism underlying mental retardation. In turn, these results may also shed light on mechanisms involved in cognitive functions.

Purification and enzymological characterization of murine neurotrypsin.

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.