Biochemical and Biophysical Research Communications, Vol.502, No.1, 98-103, 2018
Glycosylation status of nicastrin influences catalytic activity and substrate preference of gamma-secretase
gamma-Secretase complex, the assembly of nicastrin (NCT), Presenilin (PS), Presenilin Enhancer-2 (PEN-2) and Anterior pharynx defective 1 (Aph-1), catalyzes the cleavage of amyloid precursor protein to generate amyloid-beta protein (A beta), the main culprit of Alzheimer's disease. NCT becomes matured through complex glycosylation and play important role in gamma-secretase activity by interacting with catalytic subunit PS. However, the role of NCT glycosylation on gamma-secretase activity and substrate specificity is still unknown. The purpose of this study is to investigate the effect of NCT glycosylation on gamma-secretase activity and substrate specificity in a group of glycosylation mutant lectin resistant CHO (Lec) cells. CHO Lec-1 cells lack glycosyltransferase-I, GnT-I, thus N-glycan on NCT are all oligomannose type, whereas CHOLec-2 cells synthesize NCT containing sialic acid deficient oligosaccharides due to the impairment of cytidine 5'-monophosphate-sialic acid transporter. Here, we reported that mutant CHO Lec-1 and Lec-2 reduced gamma-secretase activity in both cell-based and biochemical assays, and that CHO Lec-1 preferentially reduced All A beta generation. Endogenous level of gamma-secretase complex, subcellular distribution of gamma-secretase subunits and the level of functional gamma-secretase complex remained unchanged in mutants. Interestingly, Coimmunoprecipitation study revealed that mutant gamma-secretase could recognize substrate as well as parental gamma-secretase. Our data suggests that thorough glycosylation of NCT is critical for enzymatic activity and substrate preference of gamma-secretase. (C) 2018 Elsevier Inc. All rights reserved.