Overexpression of human dynactin-associated protein (dynAP) transforms NIH3T3 cells. DynAP is a single-pass transmembrane protein with a carboxy-terminal region (amino acids 135-210) exposed to the outside of the cell possessing one potential N-glycosylation site (position 143) and a distal C-terminal region (residues 173-210) harboring a Thr/Ser-rich (T/S) cluster that may be O-glycosylated. In SDS-PAGE, dynAP migrates anomalously at ~ 45 kDa, much larger than expected (22.5 kDa) based on the amino acid composition. Using dynAP mutants, we herein showed that the T/S cluster region is responsible for the anomalous migration. The T/S cluster region is required for transport to the cytoplasmic membrane and cell transformation. We produced and purified the extracellular fragment (dynAP135-210) in secreted form and analyzed the attached glycans. Asn143 displayed complex-type glycosylation, suggesting that oligosaccharide transferase may recognize the NXT/S sequon in the secretory form, but not clearly in full-length dynAP. Core I-type O-glycosylation (Gal-GalNAc) was observed, but the mass spectrometry signal was weak, clearly indicating that further studies are needed to elucidate modifications in this region.
Keywords: Cell transformation; Dynactin-associated protein; Extracellular domain; Glycosylation; Spheroid formation; Subcellular localization.
© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.