N- and O-Glycosylation of the Intrinsically Disordered Ectodomain of PTPRA
The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we provided the first experimental evidence for its site-specific glycosylation pattern. This includes the occurrence of O-glycan at the N-glycosylation sequon among the more than 30 O-glycosylation sites confidently identified beside the 7 N-glycosylation sites. The closely spaced N- and O-glycans appear to have mutually limited the extent of further galactosylation and sialylation.
In this work, multiple LC−MS/MS analysis data sets derived from different combinations of protease and glycosidase digests of PTPRA ectodomain were analyzed in concert to build up a composite picture bottom-up. We established some ground rules governing the interpretation and correct assignment of multiple O-glycosylated peptides, particularly in confidently identifying O-glycosylation at and adjacent to the N- glycosylated sequon. We then fitted the experimentally determined site-specific glycosylation profile into the structural envelopes derived from small angle X-ray scattering (SAXS) and high-speed atomic force microscopy (HS-AFM) to build a dynamic structural model of two heavily N- and O-glycosylated PTPRA ectodomains brought together by a compact Fc domain anchor. The continuous transitioning from fully to partially extended and fold-back conformations recorded in real-time HS-AFM is provocative of how we may rethink the functional aspects of an intrinsically disordered ectodomain essentially shaped and fully shielded by glycosylation.
- Analytical Workflow:
Intact glycopeptides from all different combinations of digests were analyzed by LC−MS/MS using a HCDpdEThcD data acquisition mode and processed by Byonic. The HCD-MS2 peptide spectrum matches (PSMs) were filtered by score > 200 and PEP2D < 0.001 and then manually paired with non-filtered EThcD-MS2 PSMs identifying glycopeptides of the same m/z, sequence, and glycan within five scans. EThcD-MS2 PSMs that could not be paired were filtered by the same cutoff criteria. Site localization was manually verified based on the supporting c and z ions afforded by EThcD-MS2. Glycosylation data obtained were then used to aid structural model building of the ectodomain (ECD) based on the SEC-MALS, SAXS, and HS-AFM structural data of WT PTPRA in comparison with non-glycosylated ST2A form of PTPRA.
