Single-subunit oligosaccharyltransferases of display different and predictable peptide acceptor specificities.

causes African trypanosomiasis and contains three full-length oligosaccharyltransferase (OST) genes; two of which, STT3A and STT3B, are expressed in the bloodstream form of the parasite. These OSTs have different peptide acceptor and lipid-linked oligosaccharide donor specificities, and trypanosomes do not follow many of the canonical rules developed for other eukaryotic -glycosylation pathways, raising questions as to the basic architecture and detailed function of trypanosome OSTs. Here, we show by blue-native gel electrophoresis and stable isotope labeling in cell culture proteomics that the STT3A and STT3B proteins associate with each other in large complexes that contain no other detectable protein subunits. We probed the peptide acceptor specificities of the OSTs using a transgenic glycoprotein reporter system and performed glycoproteomics on endogenous parasite glycoproteins using sequential endoglycosidase H and peptide:-glycosidase-F digestions. This allowed us to assess the relative occupancies of numerous -glycosylation sites by endoglycosidase H-resistant -glycans originating from ManGlcNAc-PP-dolichol transferred by STT3A, and endoglycosidase H-sensitive -glycans originating from ManGlcNAc-PP-dolichol transferred by STT3B. Using machine learning, we assessed the features that best define STT3A and STT3B substrates and built an algorithm to predict the types of -glycan most likely to predominate at all the putative -glycosylation sites in the parasite proteome. Finally, molecular modeling was used to suggest why STT3A has a distinct preference for sequons containing and/or flanked by acidic amino acid residues. Together, these studies provide insights into how a highly divergent eukaryote has re-wired protein -glycosylation to provide protein sequence-specific -glycan modifications. Data are available via ProteomeXchange with identifiers PXD007236, PXD007267, and PXD007268.