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

Abstract

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.

Dissecting the Extracellular Complexity of Neuromuscular Junction Organizers

“…These account for the impressive MW of full length agrin, roughly doubling what can be estimated from the aminoacid sequence alone (∼220 kDa) (Halfter, 1993;Burg et al, 1995;Winzen et al, 2003). Interestingly, these HS/CS modifications are likely to play a functional role in agrin-mediated signaling as they were seen to modulate agrin proteolysis by the nervous system serine-protease Neurotrypsin (NT) (Reif et al, 2007;Gisler et al, 2013).…”

“…This last portion is further processed at the β site to generate 90 and 22 kDa fragments. While presented sequentially α to β, the order of cleavage in the native environment is currently unknown and could, instead, occur in parallel or in a β to α direction (Gisler et al, 2013). The exact function of the agrin cleavage products is uncertain, especially in regards to the N-terminal fragment.…”

“…Information on NT from a molecular standpoint mostly originates from three key papers published between 2007 and 2013 (Mitsui et al, 2007;Reif et al, 2008;Gisler et al, 2013). These form the basis of our biochemical understanding of NT, highlighting significant substrate specificity and optimal activity in pseudo-physiological conditions: pH∼7-8 and 100-200 mM NaCl (Mitsui et al, 2007;Reif et al, 2008).…”

“…These form the basis of our biochemical understanding of NT, highlighting significant substrate specificity and optimal activity in pseudo-physiological conditions: pH∼7-8 and 100-200 mM NaCl (Mitsui et al, 2007;Reif et al, 2008). Of particular interest is the reported modulation of NT activity by heparan sulfate (HS) glycosaminoglycans and by Ca 2+ (Reif et al, 2008;Gisler et al, 2013). Given agrin's nature as a HS proteoglycan (Halfter, 1993;Burg et al, 1995;Winzen et al, 2003) alongside its dependency on Ca 2+ for AChR clustering (Wallace, 1988), regulation of NT activity by these same factors offers an interesting point of convergence between agrin-mediated signaling and its proteolytic regulation.…”

“…As such, it would be alluring to consider the possibility of HS and Ca 2+ acting as coordinators to tie agrin cleavage to specific spatio-temporal constraints. This could not only determine selectivity and/or sequentiality of agrin α and β cleavage (Gisler et al, 2013), but also coordinate agrin processing at the NMJ to the formation of the full agrin:LRP4:MuSK complex.…”