Analysis of substrate specificity of Trypanosoma brucei oligosaccharyltransferases (OSTs) by functional expression of domain-swapped chimeras in yeast

Abstract

-Linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to the asparagine residue of a nascent polypeptide chain is catalyzed by an oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). encodes three paralogue single-protein OSTs calledSTT3A, STT3B, andSTT3C that can functionally complement the OST, making it an ideal experimental system to study the fundamental properties of OST activity. We characterized the LLO and polypeptide specificity of all threeOST isoforms and their chimeric forms in the heterologous expression host where we were able to apply yeast genetic tools and newly developed glycoproteomics methods. We demonstrated thatSTT3A accepted LLO substrates ranging from ManGlcNAc to ManGlcNAc In contrast, STT3B required more complex precursors ranging from ManGlcNAc to GlcManGlcNAc structures, and STT3C did not display any LLO preference. Sequence differences between the isoforms cluster in three distinct regions. We have swapped the individual regions between different OST proteins and identified region 2 to influence the specificity toward the LLO and region 1 to influence polypeptide substrate specificity. These results provide a basis to further investigate the molecular mechanisms and contribution of single amino acids in OST interaction with its substrates.

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

“…The results reported here and in Ref. 44 are consistent with the mechanisms of resistance in T. brucei to certain toxic lectins and carbohydrate-binding small molecules reported in an interesting series of studies ( 59 – 61 ). These workers demonstrated that the parasites could escape the effects of these trypanocidal agents, all of which bind principally to oligomannose N -glycans, by either switching to the expression of a VSG type that naturally does not carry oligomannose N -glycans or by suppressing the expression of Tb STT3B.…”

“…Although Tb STT3C (Tb927.5.910) has not been studied in this paper because it is not expressed at detectable levels in bloodstream-form T. brucei , data from its heterologous expression in yeast suggest that its peptide acceptor specificity is much more similar to Tb STT3A than Tb STT3B ( 28 , 44 ). To try to rationalize the preferences of Tb STT3A and Tb STT3C for acceptor sequons containing and/or flanked by negatively charged amino acid residues, we built molecular models of Tb STT3A, Tb STT3B, and Tb STT3C using Phyre2 ( 45 ) based on the Campylobacter lari PglB structure ( 46 ).…”

“…Such C X C sequences can have a disulfide isomerase activity ( 67 ) that might conceivably increase the acceptor substrate range of Tb STT3B and Tb STT3C. (iii) Whereas both STT3A and STT3B OSTs prefer the mature Glc 3 Man 9 GlcNAc 2 -PP-dolichol LLO donor, the T. brucei OSTs have distinct LLO donor specificities such that the presence of the ALG12-dependent c-branch of the conventional (but glucose-free) triantennary Man 9 GlcNAc 2 -PP-dolichol LLO is required by Tb STT3B but not tolerated by Tb STT3A ( 27 , 44 ). An important consequence of this differential LLO specificity is that, because Golgi mannosidase II activity is also absent in T. brucei , N -glycans derived from Tb STT3B glycosylation cannot be processed to paucimannose or complex structures, which must instead be derived exclusively from Tb STT3A glycosylation.…”

“…However, Tb STT3C is not significantly expressed in bloodstream-form or procyclic form of the parasite ( 28 , 35 , 56 – 58 ). Nevertheless, transgenic expression of Tb STT3C in S. cerevisiae clearly shows that it is a functional OST with a similar preference for sequons flanked by acidic amino acids to Tb STT3A but an LLO donor specificity like Tb STT3B ( 28 , 44 ). Amino acid sequence alignment of Tb STT3A, -B, and -C and molecular model building, based on the C. lari PglB structure ( 46 ) was performed.…”

“…Tb STT3A and Tb STT3C also contain Arg residues in place of neutral Gln-567 and Gly-406 residues in Tb STT3B, locations close enough to the active site to potentially interact with sequon-flanking anionic residues. The accompanying paper ( 44 ) elegantly addresses the issues of peptide acceptor and LLO donor specificities of all three Tb STT3s by heterologous expression of each and chimeras thereof in various yeast mutants. The accompanying paper concludes that the region containing Arg-397 and Arg-406 in Tb STT3A and Tb STT3C controls peptide acceptor specificity, and this is consistent with our suggestions from molecular modeling.…”