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

Abstract

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.

Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation

“…Teq/neurotrypsin is of particular interest as a presenilin target given its likely role in memory and cognition. Specifically, Teq mutants in Drosophila have defects in memory formation, and neurotrypsin itself has been implicated in processing of agrin, which is an important synaptic component (14,15) Finally, neurotrypsin mutations in humans cause mental retardation (16). Mechanistically, we found that in MEFs the presence of a mature ␥-secretase complex, but not the autocatalytic endoproteolysis of PSEN1 or the processing of well known ␥-secretase substrates, is required to repress neurotrypsin and agrin cleavage.…”

“…Reagents and Antibodies-Antibodies generated against agrin (R132), neurotrypsin (G95), and amyloid precursor protein-like (APPL) as well as purified Wnt3a were described previously (15,17,18). The anti-␣-tubulin (DM1A), anti-APP (22C11) to detect full-length APP, anti-PSEN1 (MAB5232), anti-GSK3␤ (07-1413), anti-actin (MAB1501), anti-H3K27me3 (07-449), anti-H3K4me3 (07-473), and anti-H3K9ac (07-352) antibodies were purchased from Millipore.…”

“…Thy-1 ) and corresponding controls were generously provided by Dr. Sonderegger (15,25). Brain samples from embryonic day 15 Psen1, and postnatal day 11 Psen2 KO mice were provided by Dr. Strooper (13) and Jayadev et al (26), respectively.…”

“…The proposed mammalian ortholog of Teq is Prss12 (also known as neurotrypsin or motopsin (14)), a serine protease recently reported to cleave the heparan sulfate proteoglycan agrin (15). To test whether neurotrypsin expression is modulated by presenilins, we assessed whether neurotrypsin is upregulated in Psen1 KO mouse brains.…”

“…Because we were able to detect neurotrypsin in cells exogenously overexpressing neurotrypsin (data not shown), we suspect that although relatively up-regulated in Psen1/2 dKO cells the levels of up-regulation are still below the detection range of our Western blot conditions. Therefore, to assess whether altering levels of neurotrypsin expression via presenilins has a functional impact, we tested presenilin-dependent effects on agrin cleavage, which is the only neurotrypsin-dependent proteolytic activity reported to date (15). Neurotrypsin cleaves agrin at two sites (␣ and ␤) to generate two fragments, ϳ90 (agrin-90) and ϳ22 kDa (agrin-22) ( Fig.…”