Characterization ofN-glycosylation and its Functional Role in SIDT1-Mediated RNA Uptake

Abstract

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multi-pass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we employ high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Our results provide crucial information about the functional impact of N-glycosylation on SIDT1. In particular, removal of the N-glycosylation from SIDT1 results in a severe defect in RNA uptake. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating RNA binding, cell surface expression and protein stability of SIDT1. Our results reveal a potential functional role for N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system, with potential implications for therapeutic applications.