Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase

Abstract

Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the WT OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, whereas mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better-quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms. Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the WT OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, whereas mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better-quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms. N-Glycosylation refers to the covalent attachment of an oligosaccharide chain on asparagine residues in proteins and is one of the most important post-translational protein modifications (1Cherepanova N. Shrimal S. Gilmore R. N-Linked glycosylation and homeostasis of the endoplasmic reticulum.Curr. Opin. Cell Biol. 2016; 41 (27085638): 57-6510.1016/j.ceb.2016.03.021Crossref PubMed Scopus (113) Google Scholar, 2Aebi M. N-Linked protein glycosylation in the ER.Biochim. Biophys. Acta. 2013; 1833 (23583305): 2430-243710.1016/j.bbamcr.2013.04.001Crossref PubMed Scopus (428) Google Scholar, 3Breitling J. Aebi M. N-Linked protein glycosylation in the endoplasmic reticulum.Cold Spring Harb. Perspect. Biol. 2013; 5 (23751184): a01335910.1101/cshperspect.a013359Crossref PubMed Scopus (153) Google Scholar). The N-glycosylation consensus, Asn-X-Ser/Thr (where X is not Pro), is the sequon, and the N-oligosaccharide chain attached to a protein is the N-glycan. The oligosaccharide donors are lipid-linked oligosaccharides (LLOs) (4Hartley M.D. Imperiali B. At the membrane frontier: a prospectus on the remarkable evolutionary conservation of polyprenols and polyprenyl-phosphates.Arch. Biochem. Biophys. 2012; 517 (22093697): 83-9710.1016/j.abb.2011.10.018Crossref PubMed Scopus (85) Google Scholar). The oligosaccharide portion of LLO is transferred to the side-chain carboxamide group of asparagine residues in the sequon the of (OST) basis of protein Biol. PubMed Scopus Google Scholar). In the yeast the transfer of the oligosaccharide on the of the endoplasmic reticulum that of the are R. N. the of protein glycosylation, as from of the Biophys. Acta. PubMed Scopus Google Scholar). The of the to be or are a for the protein in the and in the (1Cherepanova N. Shrimal S. Gilmore R. N-Linked glycosylation and homeostasis of the endoplasmic reticulum.Curr. Opin. Cell Biol. 2016; 41 (27085638): 57-6510.1016/j.ceb.2016.03.021Crossref PubMed Scopus (113) Google Scholar, 2Aebi M. N-Linked protein glycosylation in the ER.Biochim. Biophys. Acta. 2013; 1833 (23583305): 2430-243710.1016/j.bbamcr.2013.04.001Crossref PubMed Scopus (428) Google Scholar). oligosaccharide that are not attached to proteins the and the oligosaccharide protein PubMed Scopus Google Scholar). The the N-glycans from in the The N-glycans (FNGs) for of the in the yeast R. Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google Scholar). The yeast OST the of LLO The are from the to the for a and free oligosaccharide generation in Biol. PubMed Scopus Google Scholar, J. oligosaccharides to endoplasmic in Biol. PubMed Scopus Google Scholar). The of OST be using a the generation of the R. Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google Scholar). In most of the in the and the are to the activity of OST in the lumen in cells oligosaccharides are the of oligosaccharides in PubMed Scopus Google Scholar). The yeast OST is a protein of in an Stt3, and are whereas and are basis of protein Biol. PubMed Scopus Google Scholar). The Stt3 subunit the catalytic In Gilmore R. Oligosaccharyltransferase that different catalytic enzymatic PubMed Scopus Google Scholar). The Stt3 proteins from different OST for the from two and also two of and and evolutionary of the of PubMed Scopus Google Scholar). In the for the and Aebi M. in proteins for the in Biol. PubMed Google Scholar). their the and and evolutionary of the of PubMed Scopus Google Scholar, Gilmore R. of the PubMed Scopus Google Scholar). important is S. with a The protein is to the yeast Stt3 protein that the is a in and the is to the yeast Stt3 Gilmore R. of the PubMed Scopus Google Scholar). In of the Stt3, the yeast the two and two different OST containing either Ost3 or Ost6 M. R. of two either the or PubMed Scopus Google Scholar). The of the to OST complexes is R. Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google Scholar). of either Ost3 or Ost6 in of and the Ost3 and Ost6 to in yeast cells R. The from isolation of the with and of the Biol. PubMed Scopus Google Scholar). The Ost3 and Ost6 proteins a in the and the activity to the oligosaccharyl transfer reaction N. J. Aebi M. of N-linked glycosylation in yeast PubMed Scopus Google Scholar, R. Aebi M. activity of and glycosylation S. PubMed Scopus Google Scholar). the N-glycosylation to a the of the in the Ost3 and Ost6 the of R. Aebi M. activity of and glycosylation S. PubMed Scopus Google Scholar). The of the Ost3 and Ost6 In of the of yeast OST using the from the WT OST to the mutated OST in cells is the method on the N-glycosylation of the of oligosaccharyl in Biol. PubMed Scopus Google Scholar, M. N. J. M. of a catalytic motif of J. PubMed Scopus Google or the method The yeast be from PubMed Scopus Google Scholar). The is to is a is into or the different properties of the two OST complexes containing Ost3 or Ost6 in the of the the In using purified proteins with a subunit are the of the OST in vitro using proteins not the of the yeast is the epitope the of the and in the yeast for of the OST complexes and of other on the N-glycosylation of the of oligosaccharyl in Biol. PubMed Scopus Google Scholar, Gilmore R. The Stt3 protein is a subunit of the yeast and a with and Biol. PubMed Scopus Google The of to the of the in the yeast for the M. S. Aebi M. The protein is a of the yeast PubMed Scopus Google Scholar). other of epitope tag and into the of the in the yeast and the OST complexes to for enzymatic R. Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google and M. of the yeast oligosaccharyl S. PubMed Scopus Google Scholar, The of a PubMed Scopus Google Scholar, R. J. J. Aebi M. of the yeast into PubMed Scopus Google Scholar). In the of the isolation of OST complexes containing proteins is that the yeast the and and M. S. Aebi M. The protein is a of the yeast PubMed Scopus Google Scholar). the of and the membrane In the membrane the OST complexes with oligosaccharyl transfer activities M. S. Aebi M. The protein is a of the yeast PubMed Scopus Google Scholar). 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J. M. of a catalytic motif of J. PubMed Scopus Google Scholar, Gilmore R. The Stt3 protein is a subunit of the yeast and a with and Biol. PubMed Scopus Google Scholar, M. S. Aebi M. The protein is a of the yeast PubMed Scopus Google Scholar). the membrane of the OST the attachment to the be the is on the opposite of the membrane and is from the to the of the enzymatic The tag a for the M. M. J. a protein using a a from PubMed Scopus Google and the of the tag is than of other the tag of the the PubMed Scopus Google and the tag and of Biophys. Acta. 2013; PubMed Scopus Google Scholar). The and the of the OST containing the WT Stt3 subunit is important to the of the tag attachment on the enzymatic We the oligosaccharyl transfer activities the two OST complexes containing the tag on the of the Stt3 subunit and that on the of the subunit The that the tag had effects on the enzymatic We residues in the first DXD motif and the DK motif in the Stt3 as M. N. J. M. of a catalytic motif of J. PubMed Scopus Google Scholar). residues are to the peptide in the The of a PubMed Scopus Google Scholar, R. J. J. Aebi M. of the yeast into PubMed Scopus Google Scholar). We purified the OST complexes containing the WT and mutated Stt3 or and their oligosaccharyl transfer and FNG generation activities in the and absence of the peptide We also the of the mutated yeast we the Stt3 with and for in The to in a the We the N-glycosylation of the and in and is a that the of residues from the of in the is a containing N-glycosylation is one of the of the OST with a and a on the The two N-glycosylation We also the of N-glycans attached to the and the FNG and LLO in cells. we as we the different roles of the Ost3 and Ost6 The in that the and OST complexes of proteins N. J. Aebi M. of N-linked glycosylation in yeast PubMed Scopus Google Scholar, R. Aebi M. activity of and glycosylation S. PubMed Scopus Google Scholar). Here, we the different properties of the two of the OST complexes the molecular The and are Ost3 and Ost6 The OST had oligosaccharyl transfer activity than the OST The the FNG generation activity of the OST than had effects on that of the OST the Ost3 and Ost6 the catalytic activity of Stt3 in and different a or mechanism. to a that the oligosaccharyl transfer the yeast OST may not be a with the the S. Gilmore R. glycosylation is not to protein the endoplasmic reticulum in Biol. PubMed Scopus Google the Ost3 and Ost6 to with the The of a PubMed Scopus Google Scholar, R. J. J. Aebi M. of the yeast into PubMed Scopus Google Scholar). We that the different enzymatic properties of the and OST complexes in vitro will as a for future on the different roles of the in yeast cells. the two activities of the OST be mutations in the Stt3 The of and the FNG generation activity relative to the oligosaccharyl transfer activity, and the of and the FNG generation activity and are from the catalytic the the LLO activity be the peptide physiological the are in in the or of in the post-translational of the oligosaccharyl transfer In the oligosaccharyl transfer in vitro, to residues the of than residues be in in vitro to a of the OST activity the cells. We the the oligosaccharyl transfer activity in vitro and the N-glycosylation of in as an the for of the peptide in the the from a different in the Stt3 subunit The in the oligosaccharyl transfer activity in vitro mutations are with the in the N-glycosylation in cells the of N-glycans attached to the 15-residue peptide the the in vitro and in activities we peptide in the oligosaccharyl transfer the and in may be to of the with the in the OST This us to to the peptide substrate the in 5 are from the of the mutants, for a The Stt3 in a or the N-glycosylation of is to an of of that in the WT yeast cells and the In the and the of FNG OST increased and that the FNG levels in the lumen effects on yeast growth. In the content of FNG OST The that the a that a in the FNG in cells to is for yeast growth. are with the R. Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google Scholar). The FNG levels as as of the WT cells in the and the yeast growth. we not find of the FNG in the is to that the activity of LLO be a enzymatic activity the of and the of (1Cherepanova N. Shrimal S. Gilmore R. N-Linked glycosylation and homeostasis of the endoplasmic reticulum.Curr. Opin. Cell Biol. 2016; 41 (27085638): 57-6510.1016/j.ceb.2016.03.021Crossref PubMed Scopus (113) Google Scholar). The in the be for yeast in and other in and may as an In the of the the FNG the OST in the is to a in the the and R. B. of free oligosaccharides from protein N-linked glycosylation 2013; PubMed Scopus Google Scholar, J. of free oligosaccharides from the N-glycosylation S. PubMed Scopus Google Scholar). In of the two activities of a of mutations in the Stt3 subunit in vitro and in the yeast OST the different properties the and OST the mutations that the relative activity of the LLO reaction to the oligosaccharyl transfer the of peptide substrates, and the of FNG OST The of the two activities mutations useful into the generation and the physiological roles of the FNG in the in of and cells that the OST FNG in the OST not Gilmore R. N. N-glycosylation the from lipid-linked oligosaccharide S. PubMed Scopus Google Scholar). The mutations in the DXD motif and DK motif in the will be to and the the two catalytic in the OST and from The in the oligosaccharyl transfer group the peptide to the and the chain of a The from The and a peptide of yeast as an and that of yeast Stt3 M. N. J. M. of a catalytic motif of J. PubMed Scopus Google Scholar). The from The from The from The from The from in are in and tag to the the for and and in PubMed Scopus Google Scholar). The to Stt3 is epitope tag The with the epitope method to with in PubMed Scopus Google Scholar). The to is the tag and the of yeast containing a of yeast containing and on containing and in an The of in of of the glycosylation of and and the of and of Ost3 from the the effects the absence of of of Ost3 from the the effects the absence of of of Ost6 from the the effects the absence of of of Ost3 from the the effects the absence of of mutated WT The of Ost3 from the the effects the absence of The of Ost6 from the the effects the absence of in a in are in the of the tag from the M. N. J. M. of a catalytic motif of J. PubMed Scopus Google Scholar). The is a containing the and the and The and with a The of the and using from the yeast as the the of the the yeast Stt3 and the into The and the from as the a from the to the and the the and the Stt3 the of and the Ost3 and Ost6 into the of to mutations to in M. N. J. M. of a catalytic motif of J. PubMed Scopus Google of WT Stt3 the in and M. 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Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google Scholar). the of we the and The the of the OST in the of an of the peptide The method the oligosaccharide from LLO The LLO using in a for to the with The oligosaccharides with an and an and The with a of of from to of LLO from of yeast cells from a The OST purified from yeast as with modifications The of a PubMed Scopus Google Scholar). cells with in and yeast cells with and The membrane and in and a the for and the with tag with The and with and the OST with the containing peptide The using with and for protein In the proteins in transferred to as the a of and as the a of The of the an that the peptide not from the protein We that the peptide not the enzymatic cells with in and The containing from yeast cells the using The with the the OST containing from the from the yeast cells as The on using The proteins in the transferred to with the and with the The and of and The a of The with an The oligosaccharyl transfer using the peptide substrate method M. of the for the residues N-linked glycosylation PubMed Scopus Google Scholar, PubMed Google Scholar). The reaction yeast 5 substrate and purified WT or mutated OST in 5 5 and The substrate with a for The reaction for The reaction the of of The reaction with an and an and The with a of of from to for peptide of the in the of LLO to the using the The of the oligosaccharides with as with modifications J. oligosaccharides to endoplasmic in Biol. PubMed Scopus Google Scholar). The oligosaccharides with of in for the the with of in for The using a The with and with of The with of and onto the The with of to the to the The purified OST with LLO for in a reaction containing 5 5 and The than that for the oligosaccharyl transfer to for the The reaction the of of of and the reaction for for the to The with The oligosaccharides with an and an and The with a of of from to The N-glycans from from yeast as with modifications R. Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google Scholar). The yeast in yeast and the of cells and with of The in of and for for 5 the with of on for The for and the and in of containing of The for for 5 to the reaction a of for the and with The N-glycans with an and an and The with a of of from to The in the the of cells and with of The as and protein from PubMed Scopus Google Scholar). for of the to using and for protein The proteins in the transferred to with the and with the The and of and The and a of The with an The of N-glycans attached to the and the from yeast cells as with modifications R. Aebi M. free oligosaccharides Biol. 2013; PubMed Scopus Google Scholar). in the of cells and with The cells in of and of The for and on for 5 and The for and the and to The oligosaccharide on a with and on an the with The with an and an and The with a of of from to The yeast in the and with of The and of LLO as to the with The oligosaccharides with an and an and The with a of of from to The of the yeast on the of the yeast OST protein to the is a OST in the The with the The of in assessed to are the or We for the of yeast The the for the of with lipid-linked oligosaccharide endoplasmic reticulum free N-glycan a high-affinity tag

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