The Axonally Secreted Serine Proteinase Inhibitor, Neuroserpin, Inhibits Plasminogen Activators and Plasmin but Not Thrombin

Abstract

Neuroserpin is an axonally secreted serine proteinase inhibitor that is expressed in neurons during embryogenesis and in the adult nervous system. To identify target proteinases, we used a eucaryotic expression system based on the mouse myeloma cell line J558L and vectors including a promoter from an Ig--variable region, an Ig-enhancer, and the exon encoding the Igconstant region (C) and produced recombinant neuroserpin as a wild-type protein or as a fusion protein with C. We investigated the capability of recombinant neuroserpin to form SDS-stable complexes with, and to reduce the amidolytic activity of, a variety of serine proteinases in vitro. Consistent with its primary structure at the reactive site, neuroserpin exhibited inhibitory activity against trypsin-like proteinases. Although neuroserpin bound and inactivated plasminogen activators and plasmin, no interaction was observed with thrombin. A reactive site mutant of neuroserpin neither formed complexes with nor inhibited the amidolytic activity of any of the tested proteinases. Kinetic analysis of the inhibitory activity revealed neuroserpin to be a slow binding inhibitor of plasminogen activators and plasmin. Thus, we postulate that neuroserpin could represent a regulatory element of extracellular proteolytic events in the nervous system mediated by plasminogen activators or plasmin.Extracellular proteolysis exerted by serine proteinases has been implicated in a variety of processes in the nervous system during development and in adulthood. Among the serine proteinases recently reported to play a role in neural development and function, there are several well known proteins that had previously been found and characterized in nonneuronal functions, in particular blood coagulation and fibrinolysis. For example, tissue-type plasminogen activator (tPA) 1 and urokinase plasminogen activator (uPA) were found to be expressed in the nervous system (1, 2), and they have been demonstrated to be engaged in developmental processes such as cerebellar granule cell migration (3, 4), Schwann cell migration, and wrapping of axons (5), or neuromuscular synapse elimination (6). In the period of neurite outgrowth, plasminogen activators (PAs) have been found to be secreted at the growth cones of cultured neurons or neuronal cell lines (7,8), and they were demonstrated to modify the molecular composition of the neurites' substrata in vitro (9). In the adult nervous system, tPA is induced in the hippocampus after seizure, kindling, and long term potentiation (LTP) (10) and in the cerebellum after motor learning tasks (11), and mice lacking the gene for tPA (12) show a different form of hippocampal LTP (13,14). Furthermore, tPA has been demonstrated to be involved in excitotoxin-induced neuronal cell death in the murine hippocampus by converting locally secreted plasminogen to active plasmin (15, 16). Thrombin, which has been extensively characterized due to its important function in the blood clotting system, has been reported to be expressed in the nervous system (17). I...

Mutant Neuroserpin (S49P) That Causes Familial Encephalopathy with Neuroserpin Inclusion Bodies Is a Poor Proteinase Inhibitor and Readily Forms Polymers in Vitro

“…However, there is evidence that cleaved NS could result from dissociation of the complex. Osterwalder and colleagues (13,19) have shown the dissociation of the complex between tPA and chicken neuroserpin, which has Ͼ80% homology to human neuroserpin and an identical reactive center loop. Our investigations show conclusively that the generation of the reactive center loop-cleaved species of NS was mainly because of the dissociation of the NS⅐tPA complex.…”

“…Following docking, the reactive loop is cleaved, and the proteinase is inhibited by translocation over 70 Å to the lower pole of the inhibitor (16,17). The most probable target for neuroserpin is tissue plasminogen activator (tPA) (13,18,19), and it has been suggested that neuroserpintPA interactions play an important role in regulating neuronal and synaptic plasticity and memory (20 -27).…”

Mutants of Neuroserpin That Cause Dementia Accumulate as Polymers within the Endoplasmic Reticulum

“…This serpin is secreted from the axonal growth cones of the central and peripheral nervous system, where it inhibits the enzyme tissue plasminogen activator (1)(2)(3)(4)(5). The expression pattern of neuroserpin and its in vitro inhibitory activity implicate this serpin in regulating axonal growth, reducing seizure activity, controlling damage in cerebral infarction, and regulating emotional behavior and memory (6 -10).…”

An Inhibitor of Serine Proteases, Neuroserpin, Acts as a Neuroprotective Agent in a Mouse Model of Neurodegenerative Disease

“…It has been shown previously that neuroserpin can inhibit both tPA and uPA in vivo (Yepes et al, 2000) and that it is neuronally expressed and axonally secreted (Osterwalder et al, 1998). Because PAs are also secreted proteins that function in the extracellular matrix, neuroserpin provides a tool for interfering with PA activity under pathological conditions.…”