Abstract
Glycans found on receptor tyrosine kinases (RTKs) have emerged as promising targets for cancer chemotherapy, aiming to address issues such as drug resistance. However, to effectively select the target glycans, it is crucial to define the structure and function of candidate glycans in advance. Through mass spectrometric analysis, this study presents a "glycoform atlas" of epidermal growth factor receptor 2 (ErbB2), an RTK targeted for the treatment of ErbB2-positive cancers. Our analysis provides an in-depth and site-specific glycosylation profile, including both asparagine- and serine/threonine-linked glycosylation. Molecular dynamics simulations of N-glycosylated ErbB2 incorporating the identified glycan structures suggested that the N-glycan at N124 on the long flexible loop in the N-terminal region plays a role in stabilizing the ErbB2 structure. Based on the model structures obtained from the simulations, analysis employing an ErbB2 mutant deficient in N-glycosylation at N124 exhibited a significantly shorter intracellular half-life and suppressed autophosphorylation compared to wild-type ErbB2. Moreover, a structural comparison between the N-glycosylated forms of ErbB2 and its structurally homologous receptor, epidermal growth factor receptor (EGFR), demonstrated distinct variations in the distribution and density of N-glycans across these two molecules. These findings provide valuable insights into the structural and functional implications of ErbB2 glycosylation and will contribute to facilitating the establishment of glycan-targeted therapeutic strategies for ErbB2-positive cancers.