Anti-Amyloid β monoclonal antibody, clone 13C3

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.

Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic gamma-secretase inhibitors. The complexes are bibbed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.