Glycosylation Tunes Neuroserpin Physiological and Pathological Properties
Abstract
:1. Introduction
2. Results and Discussion
2.1. Expression and Purification of Glycosylated NS
2.2. gNS Is Properly Glycosylated
2.3. Conformational Analysis of gNS
2.4. gNS Inhibits tPA In Vitro
2.5. Glycosylation Reduces Heat-Induced Polymerisation of gNS
3. Materials and Methods
3.1. LEXSY Plasmid and Strain
3.2. gNS Expression and Purification
3.3. NS Expression and Purification
3.4. NS Autoproteolysis Assay
3.5. Deglycosylation Assay
3.6. MALDI-TOF MS Analysis
3.7. UHPLC-MS/MS Analysis
3.8. Circular Dichroism Analysis
3.9. Intrinsic Protein Fluorescence Assay
3.10. Polymerisation Assay
3.11. Non-Denaturating Polyacrylamide Gel Electrophoresis (PAGE)
3.12. Inhibitory Activity Test
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AC | Affinity chromatography |
CD | Circular dichroism |
Cl | Cleaved neuroserpin |
EndoH | Endoglycosidase H |
FENIB | Familial encephalopathy with neuroserpin inclusion bodies |
Frag | Proteolytic neuroserpin fragment |
gNS | Glycosylated NS |
Lat | Latent neuroserpin |
LEXSY | Leishmania expression system |
MALDI-TOF MS | Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry |
Nat | Native neuroserpin |
NS | Neuroserpin |
PNGaseF | Peptide -N-Glycosidase F |
Pol | Polymeric neuroserpin |
RCL | Reactive centre loop |
SDS-PAGE | Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis |
SEC | Size exclusion chromatography |
SERPIN | Serine Protease Inhibitor |
tPA | Tissue Plasminogen Activator |
UHPLC MS/MS | Ultra high performance liquid chromatography - tandem mass spectrometer |
References
- Miranda, E.; Lomas, D.A. Neuroserpin: A serpin to think about. Cell. Mol. Life Sci. 2006, 63, 709–722. [Google Scholar] [CrossRef]
- Whisstock, J.C.; Bottomley, S.P. Molecular gymnastics: Serpin structure, folding and misfolding. Curr. Opin. Struct. Biol. 2006, 16, 761–768. [Google Scholar] [CrossRef]
- Hastings, G.A.; Coleman, T.A.; Haudenschild, C.C.; Stefansson, S.; Smith, E.P.; Barthlow, R.; Cherry, S.; Sandkvist, M.; Lawrence, D.A. Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival. J. Biol. Chem. 1997, 272, 33062–33067. [Google Scholar] [CrossRef] [Green Version]
- Yepes, M.; Lawrence, D.A. Tissue-type plasminogen activator and neuroserpin: A well-balanced act in the nervous system? Trends Cardiovasc. Med. 2004, 14, 173–180. [Google Scholar] [CrossRef]
- Galliciotti, G.; Sonderegger, P. Neuroserpin. Front. Biosci. 2006, 11, 33–45. [Google Scholar] [CrossRef] [PubMed]
- Ricagno, S.; Caccia, S.; Sorrentino, G.; Antonini, G.; Bolognesi, M. Human neuroserpin: Structure and time-dependent inhibition. J. Mol. Biol. 2009, 388, 109–121. [Google Scholar] [CrossRef] [PubMed]
- Takehara, S.; Onda, M.; Zhang, J.; Nishiyama, M.; Yang, X.; Mikami, B.; Lomas, D.A. The 2.1-A crystal structure of native neuroserpin reveals unique structural elements that contribute to conformational instability. J. Mol. Biol. 2009, 388, 11–20. [Google Scholar] [CrossRef] [PubMed]
- Caccia, S.; Ricagno, S.; Bolognesi, M. Molecular bases of neuroserpin function and pathology. Biomol. Concepts 2010, 1, 117–130. [Google Scholar] [CrossRef]
- Gooptu, B.; Lomas, D.A. Conformational pathology of the serpins: Themes, variations, and therapeutic strategies. Annu. Rev. Biochem. 2009, 78, 147–176. [Google Scholar] [CrossRef]
- Huntington, J.A.; Read, R.J.; Carrell, R.W. Structure of a serpin-protease complex shows inhibition by deformation. Nature 2000, 407, 923–926. [Google Scholar] [CrossRef]
- Onda, M.; Belorgey, D.; Sharp, L.K.; Lomas, D.A. Latent S49P neuroserpin forms polymers in the dementia familial encephalopathy with neuroserpin inclusion bodies. J. Biol. Chem. 2005, 280, 13735–13741. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Barker-Carlson, K.; Lawrence, D.A.; Schwartz, B.S. Acyl-enzyme complexes between tissue-type plasminogen activator and neuroserpin are short-lived in vitro. J. Biol. Chem. 2002, 277, 46852–46857. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Carlson, K.-S.B.; Nguyen, L.; Schwartz, K.; Lawrence, D.A.; Schwartz, B.S. Neuroserpin Differentiates Between Forms of Tissue Type Plasminogen Activator via pH Dependent Deacylation. Front. Cell. Neurosci. 2016, 10, 154. [Google Scholar] [CrossRef] [PubMed]
- Lee, T.W.; Yang, A.S.-P.; Brittain, T.; Birch, N.P. An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: Application to neuroserpin reveals regions that differentially regulate inhibitory activity. Proteins 2015, 83, 135–152. [Google Scholar] [CrossRef] [PubMed]
- Miranda, E.; MacLeod, I.; Davies, M.J.; Pérez, J.; Römisch, K.; Crowther, D.C.; Lomas, D.A. The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB. Hum. Mol. Genet. 2008, 17, 1527–1539. [Google Scholar] [CrossRef] [Green Version]
- Davis, R.L.; Shrimpton, A.E.; Holohan, P.D.; Bradshaw, C.; Feiglin, D.; Collins, G.H.; Sonderegger, P.; Kinter, J.; Becker, L.M.; Lacbawan, F.; et al. Familial dementia caused by polymerization of mutant neuroserpin. Nature 1999, 401, 376–379. [Google Scholar] [CrossRef]
- Coutelier, M.; Andries, S.; Ghariani, S.; Dan, B.; Duyckaerts, C.; van Rijckevorsel, K.; Raftopoulos, C.; Deconinck, N.; Sonderegger, P.; Scaravilli, F.; et al. Neuroserpin Mutation Causes Electrical Status Epilepticus of Slow-Wave Sleep. Neurology 2008, 71, 64–66. [Google Scholar] [CrossRef]
- Davis, R.L.; Shrimpton, A.E.; Carrell, R.W.; Lomas, D.A.; Gerhard, L.; Baumann, B.; Lawrence, D.A.; Yepes, M.; Kim, T.S.; Ghetti, B.; et al. Association between conformational mutations in neuroserpin and onset and severity of dementia. Lancet 2002, 359, 2242–2247. [Google Scholar] [CrossRef]
- Hagen, M.C.; Murrell, J.R.; Delisle, M.-B.; Andermann, E.; Andermann, F.; Guiot, M.C.; Ghetti, B. Encephalopathy with Neuroserpin Inclusion Bodies Presenting as Progressive Myoclonus Epilepsy and Associated with a Novel Mutation in the Proteinase Inhibitor 12 Gene. Brain Pathol. 2011, 21, 575–582. [Google Scholar] [CrossRef] [Green Version]
- Davis, R.L.; Holohan, P.D.; Shrimpton, A.E.; Tatum, A.H.; Daucher, J.; Collins, G.H.; Todd, R.; Bradshaw, C.; Kent, P.; Feiglin, D.; et al. Familial encephalopathy with neuroserpin inclusion bodies. Am. J. Pathol. 1999, 155, 1901–1913. [Google Scholar] [CrossRef]
- Guadagno, N.A.; Moriconi, C.; Licursi, V.; D’Acunto, E.; Nisi, P.S.; Carucci, N.; De Jaco, A.; Cacci, E.; Negri, R.; Lupo, G.; et al. Neuroserpin polymers cause oxidative stress in a neuronal model of the dementia FENIB. Neurobiol. Dis. 2017, 103, 32–44. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lomas, D.A.; Evans, D.L.; Finch, J.T.; Carrell, R.W. The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature 1992, 357, 605–607. [Google Scholar] [CrossRef] [PubMed]
- Yamasaki, M.; Li, W.; Johnson, D.J.D.; Huntington, J.A. Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization. Nature 2008, 455, 1255–1258. [Google Scholar] [CrossRef] [PubMed]
- Ricagno, S.; Pezzullo, M.; Barbiroli, A.; Manno, M.; Levantino, M.; Santangelo, M.G.; Bonomi, F.; Bolognesi, M. Two latent and two hyperstable polymeric forms of human neuroserpin. Biophys. J. 2010, 99, 3402–3411. [Google Scholar] [CrossRef] [PubMed]
- Noto, R.; Santangelo, M.G.; Levantino, M.; Cupane, A.; Mangione, M.R.; Parisi, D.; Ricagno, S.; Bolognesi, M.; Manno, M.; Martorana, V. Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics. Biochim. Biophys. Acta 2015, 1854, 110–117. [Google Scholar] [CrossRef] [Green Version]
- Miranda, E.; Römisch, K.; Lomas, D.A. Mutants of neuroserpin that cause dementia accumulate as polymers within the endoplasmic reticulum. J. Biol. Chem. 2004, 279, 28283–28291. [Google Scholar] [CrossRef] [Green Version]
- Davies, M.J.; Miranda, E.; Roussel, B.D.; Kaufman, R.J.; Marciniak, S.J.; Lomas, D.A. Neuroserpin polymers activate NF-kappaB by a calcium signaling pathway that is independent of the unfolded protein response. J. Biol. Chem. 2009, 284, 18202–18209. [Google Scholar] [CrossRef] [Green Version]
- Kroeger, H.; Miranda, E.; MacLeod, I.; Pérez, J.; Crowther, D.C.; Marciniak, S.J.; Lomas, D.A. Endoplasmic reticulum-associated degradation (ERAD) and autophagy cooperate to degrade polymerogenic mutant serpins. J. Biol. Chem. 2009, 284, 22793–22802. [Google Scholar] [CrossRef] [Green Version]
- Schipanski, A.; Oberhauser, F.; Neumann, M.; Lange, S.; Szalay, B.; Krasemann, S.; van Leeuwen, F.W.; Galliciotti, G.; Glatzel, M. Lectin OS-9 delivers mutant neuroserpin to endoplasmic reticulum associated degradation in familial encephalopathy with neuroserpin inclusion bodies. Neurobiol. Aging 2014, 35, 2394–2403. [Google Scholar] [CrossRef]
- Moriconi, C.; Ordoñez, A.; Lupo, G.; Gooptu, B.; Irving, J.A.; Noto, R.; Martorana, V.; Manno, M.; Timpano, V.; Guadagno, N.A.; et al. Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum. FEBS J. 2015, 282, 4565–4579. [Google Scholar] [CrossRef]
- Hill, R.M.; Brennan, S.O.; Birch, N.P. Expression, purification, and functional characterization of the serine protease inhibitor neuroserpin expressed in Drosophila S2 cells. Protein Expr. Purif. 2001, 22, 406–413. [Google Scholar] [CrossRef] [PubMed]
- Belorgey, D.; Sharp, L.K.; Crowther, D.C.; Onda, M.; Johansson, J.; Lomas, D.A. Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers. Eur. J. Biochem. 2004, 271, 3360–3367. [Google Scholar] [CrossRef] [PubMed]
- Breitling, R.; Klingner, S.; Callewaert, N.; Pietrucha, R.; Geyer, A.; Ehrlich, G.; Hartung, R.; Müller, A.; Contreras, R.; Beverley, S.M.; et al. Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr. Purif. 2002, 25, 209–218. [Google Scholar] [CrossRef]
- Klatt, S.; Konthur, Z. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae. Microb. Cell Factories 2012, 11, 97. [Google Scholar] [CrossRef] [Green Version]
- Trimble, R.B.; Tarentino, A.L. Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: Endo F1, endo F2, and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans. J. Biol. Chem. 1991, 266, 1646–1651. [Google Scholar]
- Noto, R.; Santangelo, M.G.; Ricagno, S.; Mangione, M.R.; Levantino, M.; Pezzullo, M.; Martorana, V.; Cupane, A.; Bolognesi, M.; Manno, M. The tempered polymerization of human neuroserpin. PLoS ONE 2012, 7, e32444. [Google Scholar] [CrossRef]
- Galliciotti, G.; Glatzel, M.; Kinter, J.; Kozlov, S.V.; Cinelli, P.; Rülicke, T.; Sonderegger, P. Accumulation of mutant neuroserpin precedes development of clinical symptoms in familial encephalopathy with neuroserpin inclusion bodies. Am. J. Pathol. 2007, 170, 1305–1313. [Google Scholar] [CrossRef] [Green Version]
- Tedeschi, G.; Pagliato, L.; Negroni, M.; Montorfano, G.; Corsetto, P.; Nonnis, S.; Negri, A.; Rizzo, A.M. Protein pattern of Xenopus laevis embryos grown in simulated microgravity. Cell Biol. Int. 2011, 35, 249–258. [Google Scholar] [CrossRef]
- Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J.V.; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2006, 1, 2856–2860. [Google Scholar] [CrossRef]
- Yu, Q.; Wang, B.; Chen, Z.; Urabe, G.; Glover, M.S.; Shi, X.; Guo, L.W.; Kent, K.C.; Li, L. Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization. J. Am. Soc. Mass Spectrom. 2017, 28, 1751–1764. [Google Scholar] [CrossRef]
- Irving, J.A.; Ekeowa, U.I.; Belorgey, D.; Haq, I.; Gooptu, B.; Miranda, E.; Pérez, J.; Roussel, B.D.; Ordóñez, A.; Dalton, L.E.; et al. The serpinopathies studying serpin polymerization in vivo. Methods Enzymol. 2011, 501, 421–466. [Google Scholar] [CrossRef] [PubMed]
- Saga, G.; Sessa, F.; Barbiroli, A.; Santambrogio, C.; Russo, R.; Sala, M.; Raccosta, S.; Martorana, V.; Caccia, S.; Noto, R.; et al. Embelin binds to human neuroserpin and impairs its polymerisation. Sci. Rep. 2016, 6, 769. [Google Scholar] [CrossRef] [PubMed] [Green Version]
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Visentin, C.; Broggini, L.; Sala, B.M.; Russo, R.; Barbiroli, A.; Santambrogio, C.; Nonnis, S.; Dubnovitsky, A.; Bolognesi, M.; Miranda, E.; et al. Glycosylation Tunes Neuroserpin Physiological and Pathological Properties. Int. J. Mol. Sci. 2020, 21, 3235. https://doi.org/10.3390/ijms21093235
Visentin C, Broggini L, Sala BM, Russo R, Barbiroli A, Santambrogio C, Nonnis S, Dubnovitsky A, Bolognesi M, Miranda E, et al. Glycosylation Tunes Neuroserpin Physiological and Pathological Properties. International Journal of Molecular Sciences. 2020; 21(9):3235. https://doi.org/10.3390/ijms21093235
Chicago/Turabian StyleVisentin, Cristina, Luca Broggini, Benedetta Maria Sala, Rosaria Russo, Alberto Barbiroli, Carlo Santambrogio, Simona Nonnis, Anatoly Dubnovitsky, Martino Bolognesi, Elena Miranda, and et al. 2020. "Glycosylation Tunes Neuroserpin Physiological and Pathological Properties" International Journal of Molecular Sciences 21, no. 9: 3235. https://doi.org/10.3390/ijms21093235