title: Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum.
abstract: The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site-directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early-onset dementia. Our findings indicate that N401 glycosylation reports lability of the C-terminal end of neuroserpin in its native state. This C-terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant-specific N-linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.
authors.affiliation: Department of Biology and Biotechnologies 'Charles Darwin', Sapienza University of Rome, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Moriconi
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, UK.
authors.forename: Adriana
authors.initials: A
authors.lastname: Ordoñez
authors.affiliation: Department of Chemistry, Sapienza University of Rome, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Lupo
authors.affiliation: Division of Asthma, Allergy and Lung Biology, King's College London, UK.
authors.forename: Bibek
authors.initials: B
authors.lastname: Gooptu
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Irving
authors.affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
authors.forename: Rosina
authors.initials: R
authors.lastname: Noto
authors.affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Martorana
authors.affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Manno
authors.affiliation: Department of Biology and Biotechnologies 'Charles Darwin', Sapienza University of Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Timpano
authors.affiliation: Department of Biology and Biotechnologies 'Charles Darwin', Sapienza University of Rome, Italy.
authors.forename: Noemi A
authors.initials: NA
authors.lastname: Guadagno
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, UK.
authors.forename: Lucy
authors.initials: L
authors.lastname: Dalton
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, UK.
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Marciniak
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, UK.
authors.forename: David A
authors.initials: DA
authors.lastname: Lomas
authors.affiliation: Department of Biology and Biotechnologies 'Charles Darwin', Sapienza University of Rome, Italy.|Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Italy.
authors.forename: Elena
authors.identifier: http://orcid.org/0000-0002-0586-8795
authors.initials: E
authors.lastname: Miranda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D058105:Polymerization; D051381:Rats; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D000097522:Neuroserpin
chemical_list: D001704:Biopolymers; D009479:Neuropeptides; D015843:Serpins; D000097522:Neuroserpin
keywords: conformational disease; neurodegeneration; protein aggregation; serpin; serpinopathies