دورية أكاديمية
أعرض في EDS

N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.

التفاصيل البيبلوغرافية
العنوان: N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.
المؤلفون: Brockmöller S; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany. Sabrina.Brockmoeller@yahoo.de., Molitor LM; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany., Seeger T; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany., Worek F; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany., Rothmiller S; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.
المصدر: The Journal of membrane biology [J Membr Biol] 2024 Aug; Vol. 257 (3-4), pp. 245-256. Date of Electronic Publication: 2024 Jul 05.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: Springer Country of Publication: United States NLM ID: 0211301 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1432-1424 (Electronic) Linking ISSN: 00222631 NLM ISO Abbreviation: J Membr Biol Subsets: MEDLINE
أسماء مطبوعة: Original Publication: New York, Springer.
مواضيع طبية MeSH: alpha7 Nicotinic Acetylcholine Receptor*/genetics , alpha7 Nicotinic Acetylcholine Receptor*/metabolism , Cricetulus*, Animals ; CHO Cells ; Glycosylation ; Humans ; Cricetinae ; Transgenes ; Molecular Chaperones/genetics ; Molecular Chaperones/metabolism ; Cell Survival/genetics ; Intracellular Signaling Peptides and Proteins
مستخلص: The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.
(© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
References: Adams BM, Canniff NP, Guay KP, Hebert DN (2021) The role of endoplasmic reticulum chaperones in protein folding and quality control. Prog Mol Subcell Biol 59:27–50. (PMID: 340508619185992)
Akaike A, Shimohama S, Misu Y (eds) (2018) Nicotinic acetylcholine receptor signaling in neuroprotection. Singapore. https://doi.org/10.1007/978-981-10-8488-1.
Brockmöller S, Seeger T, Worek F, Rothmiller S (2023) Recombinant cellular model system for human muscle-type nicotinic acetylcholine receptor α12β1δε. Cell Stress Chaperones 28(6):1013–1025. (PMID: 3800656510746606)
Caramelo JJ, Parodi AJ (2008) Getting in and out from calnexin/calreticulin cycles. J Biol Chem 283(16):10221–10225. (PMID: 183030192447651)
Castillo M, Mulet J, Gutiérrez LM, Ortiz JA, Castelán F, Gerber S, Sala S, Sala F, Criado M (2005) Dual role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors. J Biol Chem 280(29):27062–27068. (PMID: 15927954)
Chen D, Dang H, Patrick JW (1998) Contributions of N-linked glycosylation to the expression of a functional α7-nicotinic receptor in Xenopus oocytes. J Neurochem 70(1):349–357. (PMID: 9422381)
Delacour D, Cramm-Behrens CI, Drobecq H, Le Bivic A, Naim HY, Jacob R (2006) Requirement for galectin-3 in apical protein sorting. Curr Biol 16(4):408–414. (PMID: 16488876)
Dellisanti CD, Yao Y, Stroud JC, Wang Z-Z, Chen L (2007) Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution. Nat Neurosci 10(8):953–962. (PMID: 17643119)
Elgoyhen AB, Johnson DS, Boulter J, Vetter DE, Heinemann S (1994) α9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 79(4):705–715. (PMID: 7954834)
Gehle VM, Walcott EC, Nishizaki T, Sumikawa K (1997) N-glycosylation at the conserved sites ensures the expression of properly folded functional ACh receptors. Mol Brain Res 45(2):219–229. (PMID: 9149096)
Green WN (1999) Ion channel assembly: creating structures that function. J Gen Physiol 113(2):163–170. (PMID: 99258152223360)
Gu Y, Hall ZW (1988) Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle. J Biol Chem 263(26):12878–12885. (PMID: 3417640)
Gu Y, Ralston E, Murphy-Erdosh C, Black RA, Hall ZW (1989) Acetylcholine receptor in a C2 muscle cell variant is retained in the endoplasmic reticulum. J Cell Biol 109(2):729–738. (PMID: 2668304)
Gu S, Matta JA, Lord B, Harrington AW, Sutton SW, Davini WB, Bredt DS (2016) Brain α7 nicotinic acetylcholine receptor assembly requires NACHO. Neuron 89(5):948–955. (PMID: 26875622)
Guha P, Bandyopadhyaya G, Polumuri SK, Chumsri S, Gade P, Kalvakolanu DV, Ahmed H (2014) Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, α9 nicotinic acetylcholine receptor and STAT3. Breast Cancer Res Treat 145(1):5–22. (PMID: 246685004028025)
Huang S, Li S-X, Bren N, Cheng K, Gomoto R, Chen L, Sine SM (2013) Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera. Biochem J 454(2):303–310. (PMID: 23800261)
Keller SH, Taylor P (1999) Determinants responsible for assembly of the nicotinic acetylcholine receptor. J Gen Physiol 113(2):171–176. (PMID: 99258162223362)
Keller SH, Lindstrom J, Taylor P (1998) Inhibition of glucose trimming with castanospermine reduces calnexin association and promotes proteasome degradation of the alpha-subunit of the nicotinic acetylcholine receptor. J Biol Chem 273(27):17064–17072. (PMID: 9642271)
Kweon H-J, Gu S, Witham E, Dhara M, Yu H, Mandon ED, Jawhari A, Bredt DS (2020) NACHO engages N-glycosylation ER chaperone pathways for α7 nicotinic receptor assembly. Cell Rep 32(6):108025. https://doi.org/10.1016/j.celrep.2020.108025. (PMID: 10.1016/j.celrep.2020.10802532783947)
Lansdell SJ, Gee VJ, Harkness PC, Doward AI, Baker ER, Gibb AJ, Millar NS (2005) RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol Pharmacol 68(5):1431–1438. (PMID: 16120769)
Loring RH (2022) Speculation on how RIC-3 and other chaperones facilitate α7 nicotinic receptor folding and assembly. Molecules 27(14):4527. https://doi.org/10.3390/molecules27144527. (PMID: 10.3390/molecules27144527358894009318448)
Marinko JT, Huang H, Penn WD, Capra JA, Schlebach JP, Sanders CR (2019) Folding and misfolding of human membrane proteins in health and disease: from single molecules to cellular proteostasis. Chem Rev 119(9):5537–5606. (PMID: 306086666506414)
Martin PT, Sanes JR (1995) Role for a synapse-specific carbohydrate in agrin-induced clustering of acetylcholine receptors. Neuron 14(4):743–754. (PMID: 7718237)
Matta JA, Gu S, Davini WB, Lord B, Siuda ER, Harrington AW, Bredt DS (2017) NACHO mediates nicotinic acetylcholine receptor function throughout the brain. Cell Rep 19(4):688–696. (PMID: 28445721)
Merlie JP, Lindstrom J (1983) Assembly in vivo of mouse muscle acetylcholine receptor: identification of an α subunit species that may be an assembly intermediate. Cell 34(3):747–757. (PMID: 6627392)
Millar NS (2008) RIC-3: a nicotinic acetylcholine receptor chaperone. Br J Pharmacol 153(S1):177–183.
Millar NS, Harkness PC (2008) Assembly and trafficking of nicotinic acetylcholine receptors (Review). Mol Membr Biol 25(4):279–292. (PMID: 18446614)
Mohanty S, Chaudhary BP, Zoetewey D (2020) Structural insight into the mechanism of N-linked glycosylation by oligosaccharyltransferase. Biomolecules 10(4):624. https://doi.org/10.3390/biom10040624. (PMID: 10.3390/biom10040624323166037226087)
Murray TA, Liu Q, Whiteaker P, Wu J, Lukas RJ (2009) Nicotinic acetylcholine receptor alpha7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors. Acta Pharmacol Sin 30(6):828–841. (PMID: 194984234002364)
Nakagawa F, Schulte BA, Wu JY, Spicer SS (1986) GABAergic neurons of rodent brain correspond partially with those staining for glycoconjugate with terminal N-acetylgalactosamine. J Neurocytol 15:389–396. (PMID: 2427661)
Obergrussberger A, Haarmann C, Rinke I, Becker N, Guinot D, Brueggemann A, Stoelzle-Feix S, George M, Fertig N (2014) Automated patch clamp analysis of nAChα7 and Nav1. 7 channels. Curr Protocols Pharmacol 65(1):11–13.
Poulter L, Burlingame AL (1990) Desorption mass spectrometry of oligosaccharides coupled with hydrophobic chromophores. In: Methods in enzymology, vol. 193. Academic Press, pp 661–689.
Roth FC, Numberger M, Draguhn A (2023) Patch-clamp-technik. Springer, Berlin, Heidelberg.
Rudell JC, Borges LS, Rudell JB, Beck KA, Ferns MJ (2014) Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor. J Biol Chem 289(1):203–214. (PMID: 24240098)
Rudell JC, Borges LS, Yarov-Yarovoy V, Ferns M (2020) The MX-Helix of muscle nAChR subunits regulates receptor assembly and surface trafficking. Front Mol Neurosci 13:48. (PMID: 322656537105636)
Sanes JR, Cheney JM (1982) Lectin binding reveals a synapse-specific carbohydrate in skeletal muscle. Nature 300(5893):646–647. (PMID: 7144916)
Sanes JR, Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22(1):389–442. (PMID: 10202544)
Scott LJ, Bacou F, Sanes JR (1988) A synapse-specific carbohydrate at the neuromuscular junction: association with both acetylcholinesterase and a glycolipid. J Neurosci 8(3):932–944. (PMID: 33467306569238)
Shoji H, Takahashi N, Nomoto H, Ishikawa M, Shimada I, Arata Y, Hayashi K (1992) Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica. Eur J Biochem 207(2):631–641. (PMID: 1633814)
Shurer CR, Kuo JC-H, Roberts LM, Gandhi JG, Colville MJ, Enoki TA, Pan H, Su J, Noble JM, Hollander MJ, O’Donnell JP, Yin R, Pedram K, Möckl L, Kourkoutis LF, Moerner WE, Bertozzi CR, Feigenson GW, Reesink HL, Paszek MJ (2019) Physical principles of membrane shape regulation by the glycocalyx. Cell 177(7):1757-1770.e21. (PMID: 310562826768631)
Sine SM, Strikwerda JR, Mazzaferro S (2019) Structural basis for α-bungarotoxin insensitivity of neuronal nicotinic acetylcholine receptors. Neuropharmacology 160:107660. (PMID: 311631796842095)
Sweileh W, Wenberg K, Xu J, Forsayeth J, Hardy S, Loring RH (2000) Multistep expression and assembly of neuronal nicotinic receptors is both host-cell-and receptor-subtype-dependent. Mol Brain Res 75(2):293–302. (PMID: 10686351)
Vagin O, Kraut JA, Sachs G (2009) Role of N-glycosylation in trafficking of apical membrane proteins in epithelia. Am J Physiol Renal Physiol 296(3):F459–F469. (PMID: 18971212)
Wanamaker CP, Christianson JC, Green WN (2003) Regulation of nicotinic acetylcholine receptor assembly. Ann NY Acad Sci 998(1):66–80. (PMID: 14592864)
Wessler I, Kirkpatrick CJ (2008) Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans. Br J Pharmacol 154(8):1558–1571. (PMID: 185003662518461)
Williams ME, Burton B, Urrutia A, Shcherbatko A, Chavez-Noriega LE, Cohen CJ, Aiyar J (2005) Ric-3 promotes functional expression of the nicotinic acetylcholine receptor alpha7 subunit in mammalian cells. J Biol Chem 280(2):1257–1263. (PMID: 15504725)
فهرسة مساهمة: Keywords: N-Glycosylation; NACHO; Nicotinic acetylcholine receptor; RIC3
المشرفين على المادة: 0 (alpha7 Nicotinic Acetylcholine Receptor)
0 (RIC3 protein, human)
0 (Molecular Chaperones)
0 (Intracellular Signaling Peptides and Proteins)
تواريخ الأحداث: Date Created: 20240705 Date Completed: 20240730 Latest Revision: 20240730
رمز التحديث: 20240730
DOI: 10.1007/s00232-024-00317-0
PMID: 38967800
قاعدة البيانات: MEDLINE
الوصف
تدمد:1432-1424
DOI:10.1007/s00232-024-00317-0