Point Mutations at N-linked Glycosylation Sites of Human Scavenger Receptor AI Reduce Acetyl-Low Density Lipoprotein and Oligomeric Amyloid- Internalization through Attenuating Translation Efficiency

• Main Authors: Yun-Hao Lee, 李昀澔
• Other Authors: Huey-Jen Tsay
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/m99ju7

碩士 === 國立陽明大學 === 神經科學研究所 === 100 === The insufficient clearance of oligomeric amyloid  (oA) in the early stages of Alzheimer's disease (AD) leads to the synaptic damage and cognitive deficits. Previously, we have identified scavenger receptor A (SR-A) as a prominent subtype of scavenger receptor family for oA internalization by naïve primary microglia. The status of N-linked glycosylation is relevant to surface expression and ligand binding in class B scavenger receptor SR-BI and CD36. This study aims at identifying the influence of N-linked glycosylation on structure and function in SR-AI. There are seven predicted N-linked glycosylation sites at N82, N102, N143, N184, N221, N249, and N267, respectively. Here, we constructed single, double, triple, quintuple, and septuple point mutations of human SR-AI at the seven sites and overexpressed the mutants in COS-7 cells. The cells were treated with Fluorescein amidite (FAM)-labeled oA and Alexa 488-labeled acetylated low density lipoprotein (AcLDL) to examine the ability of ligand internalization. The live immunostaining and Western blot analysis of biotinylated membrane proteins were performed to examine the level of surface-targeted SR-AI. The N-linked glycosylation site at N102, N143 and N184 are glycosylated. The double mutants N1,4-7 and N1,2,5-7 and triple mutant N1,5-7 showed reduced ligand internalization compared to wild-type SR-AI. The surface-targeted SR-A and internalized ligand of quintuple mutants N5,6 and N1,7 were even lower than that of triple mutants and that of septuple mutant were barely detected. Our data are consistent with the previous study of SR-BI and CD36 which indicate that mutations reduce surface-targeting of receptor. This study suggests that surface targeting and ligand internalization of SR-AI are down-regulated by the absence of N-linked glycosylation at N102, 143 and 184 or the reduced translational efficiency induced by point mutations.