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

Abstract

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.