High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha

The intracellular phosphatase domain of the receptor-typeproteintyrosine phosphatase alpha (PTPRA) is known to regulate various signalingpathways related to cell adhesion through c-Src kinase activation.In contrast, the functional significance of its relatively short,intrinsically disordered, and heavily glycosylated ectodomain remainsunclear. Through detailed mass spectrometry analyses of a combinationof protease and glycosidase digests, we now provide the first experimentalevidence for its site-specific glycosylation pattern. This includesthe occurrence of O-glycan at the N-glycosylationsequon among the more than 30 O-glycosylation sites confidently identifiedbeside the 7 N-glycosylation sites. The closely spaced N- and O-glycansappear to have mutually limited the extent of further galactosylationand sialylation. An immature smaller form of full-length PTPRA wasfound to be deficient in O-glycosylation, most likely due to failureto transit the Golgi. N-glycosylation, on the other hand, is dispensablefor cell surface expression and contributes less than the extensiveO-glycosylation to the overall solution structure of the ectodomain.The glycosylation information is combined with the overall structuralfeatures of the ectodomain derived from small-angle X-ray scatteringand high-speed atomic force microscopy monitoring to establish a dynamicstructural model of the densely glycosylated PTPRA ectodomain. Theobserved high structural flexibility, as manifested by continuoustransitioning from fully to partially extended and fold-back conformations,suggests that the receptor-type phosphatase is anchored to the membraneand kept mostly at a monomeric state through an ectodomain shapedand fully shielded by glycosylation.