Purification and enzymological characterization of murine neurotrypsin

Abstract

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.

Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction

“…S9, none of these proteins was cleaved by neurotrypsin. These results corroborated previous indications of a highly selective proteolytic activity of neurotrypsin versus agrin (Reif et al, 2007;Reif et al, 2008).…”

“…5A). Detailed characterizations of the individual cleavage sites had previously demonstrated that substitution of the basic P1 amino acid by alanine prevented cleavage by neurotrypsin at each site (Reif et al, 2007;Reif et al, 2008). Coexpression tests in HeLa cells confirmed the effect of these mutations by showing the resistance of double-mutated full-length agrin against proteolytic cleavage by neurotrypsin (supplementary material Fig.…”

“…S10) of neurotrypsin-deficient mice. The enzymatic characteristics of the postulated protease should be very similar to neurotrypsin, because its proteolytic function should also depend on the presence of the P1 basic residue at each cleavage site of agrin (Reif et al, 2007;Reif et al, 2008). An isoenzyme of neurotrypsin with a different expression pattern can be excluded, because only one neurotrypsin gene is found in the murine genome.…”

Injection of a Soluble Fragment of Neural Agrin (NT-1654) Considerably Improves the Muscle Pathology Caused by the Disassembly of the Neuromuscular Junction

“…The protein starts with L1519 (A2ASQ1 (AGRIN_MOUSE), UniProtKB/Swiss-Prot) and ends with the natural C-terminus of agrin. The fragment contains the z-8 amino acid insert and in addition the lysine to alanine mutation which renders the protein protected against site specific neurotrypsin cleavage at the β-cleavage site [36]. NT-1654 was transiently expressed in CHO-S cells in suspension culture (Evitria, Switzerland) to a level of up to 900 mg/L.…”

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

“…Neurotrypsin cleaves agrin at two homologous conserved sites (α-and β-site), resulting in an N-terminal part, a middle 90-kDa fragment (agrin-90) and a C-terminal 22-kDa fragment (agrin-22). However, short peptides only consisting of the amino acid sequence of the αor β-site are not cleaved by neurotrypsin, indicating that the specificity of neurotrypsin for agrin is further enhanced by exosite interactions [4,7]. However, short peptides only consisting of the amino acid sequence of the αor β-site are not cleaved by neurotrypsin, indicating that the specificity of neurotrypsin for agrin is further enhanced by exosite interactions [4,7].…”