Ricerc@Sapienza

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.