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.

Keywords: glycosylation; neuroserpin; protein polymerisation.

Figure 1

Human glycosylated NS (gNS) expression and purification in LEXSY. (A) Primary sequence of the construct used for gNS expression in LEXSY (UniProtKB - Q99574). The signal peptide sequence is colored in blue, the HisTag is green and the spacer in grey. The neuroserpin (NS) sequence is black, the glycosylation sites are in red and the triangle highlights the protease cleavage site. (B) SDS-PAGE analysis of gNS purification after AC and SEC chromatographic steps according to the protocol shown in Figure S1A. (C) SDS-PAGE analysis of optimized gNS purification after the AC and SEC chromatographic steps (see also Figure S1B). Abbreviations: Nat: native; CL: cleaved; AC: affinity chromatography; SEC: size exclusion chromatography.

Figure 2

Assessment of N-glycosylation. (A) SDS-PAGE analysis of the enzymatic deglycosylation of gNS using EndoH or PNGaseF deglycosylases. All samples are a mixture of native and cleaved forms. Black and white arrows refer to lane 1 (gNS) and lane 2 (PNGaseF), respectively. (B,C) Matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) MS spectra of NS (B) and gNS. Abbreviations: Nat: native; Nat*: deglycosylated Nat; Cl: cleaved; Cl*: deglycosylated Cl.

Figure 3

Identification of N-glycosylation sites. UHPLC-MS/MS analysis of glycopeptides WVENNTNNLVK (A,C) and DANLTGLSDNKEIFLSK (B,D). The peptides are detected in the MS spectrum as 2+ ion with m/z = 767.3906 (A) and 3+ ion with m/z = 757.7227 (B). These signals are selected and used as parent ions (PIs) for higher energy collision dissociation (HCD) fragmentation (C,D). Two glyco-diagnostic peaks are present in the fragmentation spectra (HexNAc, m/z 204.09; HexNAc fragment, m/z 126.05 Da), indicating that PIs are glycopeptides. Identified a-, b- and y-fragment ions are labelled (*, loss of HexNAc during fragmentation; #, loss of HexNAc2 during fragmentation).

Figure 4

Conformational characterisation of gNS. (A) Far-UV circular dichroism (CD) spectra of native gNS and NS recorded at 20 °C. (B,C) Temperature ramp of NS (B) analogously reported in [24] and gNS (C) performed recording ellipticity at 216 nm during heating from 20 to 95 °C (black) and cooling down (grey). The first derivative is reported in the bottom (blue) and the two transitions T1 and T2 are highlighted by the red arrows. (D) Far-UV CD spectra of gNS recorded at 20 °C before (black) or after (green) heating the sample to 95 °C. (E) Intrinsic tryptophan fluorescence of native (solid line) and polymeric (dashed line) gNS recorded at 20 °C. The buffer 20 mM Tris-HCl pH 8, 50 mM KCl was used in all the measurements.

Figure 5

Comparison of tissue plasminogen activator (tPA) inhibition by NS and gNS. (A) tPA activity tests in the absence (red) and presence of 60 nM of NS (blue), native (green) or incubated for 24 h at 45 °C (grey) gNS. (B) The ability of gNS and NS to inhibit tPA at two different concentrations (75 and 150 nM) was compared. (C) The inhibitory efficiency of gNS or NS is reported in comparison to the control reaction. The activity of tPA in the positive control after 56 h is set to 100% and all other values have been normalised accordingly. In the control reaction, no gNS or NS was added.

Figure 6

Heat-induced polymerisation of gNS. A solution of gNS or NS was incubated at 45 °C and analysed at different time points. SEC analysis of NS (A) or gNS (B) using an Increase Superdex 200 column. Aliquots of NS or gNS were analysed by non-denaturing (C) and denaturing (D) PAGE. Abbreviations: Nat: native; Cl: cleaved; Lat: latent; Pol: polymers.