Glycosylation Tunes Neuroserpin Physiological and Pathological Properties

Abstract

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Iripin-3, a New Salivary Protein Isolated From Ixodes ricinus Ticks, Displays Immunomodulatory and Anti-Hemostatic Properties In Vitro

“…However, recombinant Iripin-3 was prepared in an E. coli expression system, and therefore it lacks glycosylation. Glycosylation has been shown to reduce the propensity of serpins for polymerization ( 122 ) and increase the stability and half-life of circulating serpins by conferring resistance to proteolytic degradation ( 123 , 124 ). The impact of glycosylation on the biological function of serpins is less clear.…”

Neuroserpin: structure, function, physiology and pathology

“…A detailed study has also shown that pH has an important role in substrate recognition and deacylation rates during neuroserpin’s inhibition of tPA, which is different between the single-chain and two-chain forms of tPA [ 40 ]. It should be noted that many of these studies have been performed using recombinant neuroserpin lacking N -glycans, but a recent report shows that N-glycosylation slightly improves the inhibitory activity of neuroserpin against tPA [ 41 ].…”

“…Most of the in vitro studies about conformational stability and polymerisation of neuroserpin have been performed using bacterial recombinant protein, thus overlooking the effects of glycosylation. Recently, a new expression model based on a modified Leishmania strain has been set up to produce neuroserpin with mammalian-like glycosylation [ 41 ]. This in vitro work has confirmed a decreased tendency to polymerisation, a higher propensity to acquire the latent conformation, and a slight increase in inhibitory activity for the glycosylated protein.…”

Embelin as Lead Compound for New Neuroserpin Polymerization Inhibitors

“…NS, as many secreted proteins, is glycosylated, presenting two N-glycosylation chains in positions N157 and N321 that decrease spontaneous polymerization in vitro and in vivo [ 17 , 18 ]. Indeed, alteration of the glycosylation pattern due to their removal or pathologic mutation leads to enhanced polymers (Pol) formation [ 17 , 18 ]. The molecular structure of serpin Pol is still under debate and several models have been proposed, mainly for alfa-1 antitrypsin and alfa-1 antithrombin, two prototypical serpins for molecular studies.…”

“…NS, as many secreted proteins, is glycosylated, presenting two N-glycosylation chains in positions N157 and N321 that decrease spontaneous polymerization in vitro and in vivo [17,18]. Indeed, alteration of the glycosylation pattern due to their removal or pathologic mutation leads to enhanced polymers (Pol) formation [17,18].…”