Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100809
ISSN1083-351X
AutoresJillianne Eyring, Chia‐Wei Lin, Elsy M. Ngwa, Jérémy Boilevin, Giorgio Pesciullesi, Kaspar P. Locher, Tamis Darbre, Jean‐Louis Reymond, Markus Aebi,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoOligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits. Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits. Asparagine-linked glycosylation (N-glycosylation) of proteins is one of the most common covalent posttranslational protein modifications in eukaryotes. Homologous processes are found in archaea and bacteria (1Schwarz F. Aebi M. Mechanisms and principles of N-linked protein glycosylation.Curr. Opin. Struct. Biol. 2011; 21: 576-582Crossref PubMed Scopus (438) Google Scholar). The N-linked glycans fulfill a multitude of functions, such as regulating and controlling protein folding and intracellular trafficking or defining interactions at the cell surface (2Helenius A. Aebi M. Roles of N-linked glycans in the endoplasmic reticulum.Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1533) Google Scholar, 3Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: Diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (962) Google Scholar, 4Varki A. Biological roles of glycans.Glycobiology. 2017; 27: 3-49Crossref PubMed Scopus (861) Google Scholar). In a key step of this pathway, the oligosaccharyltransferase (OST) enzyme transfers a preassembled oligosaccharide from a lipid carrier onto an asparagine residue on proteins in the endoplasmic reticulum (ER) (5Breitling J. Aebi M. N-linked protein glycosylation in the endoplasmic reticulum.Cold Spring Harb. Perspect. Biol. 2013; 5: a013359Crossref PubMed Scopus (140) Google Scholar). The modified asparagine residue is part of the consensus sequon N-X-(S/T). OST binds both the acceptor protein and the donor lipid-linked oligosaccharide (LLO) and catalyzes the formation of a glycosidic bond between the amide nitrogen of the asparagine side chain and the C1 carbon of the reducing end N-acetylglucosamine (GlcNAc) residue of the oligosaccharide. High-resolution structures and biochemical studies on the bacterial OST, PglB from Campylobacter lari, propose mechanisms for peptide and LLO binding, amide activation, and catalysis (6Lizak C. Gerber S. Numao S. Aebi M. Locher K.P. X-ray structure of a bacterial oligosaccharyltransferase.Nature. 2011; 474: 350-355Crossref PubMed Scopus (258) Google Scholar, 7Gerber S. Lizak C. Michaud G. Bucher M. Darbre T. Aebi M. Reymond J.L. Locher K.P. Mechanism of bacterial oligosaccharyltransferase: In vitro quantification of sequon binding and catalysis.J. Biol. Chem. 2013; 288: 8849-8861Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Lizak C. Gerber S. Michaud G. Schubert M. Fan Y.Y. Bucher M. Darbre T. Aebi M. Reymond J.L. Locher K.P. Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation.Nat. Commun. 2013; 4: 2627Crossref PubMed Scopus (42) Google Scholar, 9Lizak C. Gerber S. Zinne D. Michaud G. Schubert M. Chen F. Bucher M. Darbre T. Zenobi R. Reymond J.L. Locher K.P. A catalytically essential motif in external loop 5 of the bacterial oligosaccharyltransferase PglB.J. Biol. Chem. 2014; 289: 735-746Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 10Napiorkowska M. Boilevin J. Sovdat T. Darbre T. Reymond J.L. Aebi M. Locher K.P. Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase.Nat. Struct. Mol. Biol. 2017; 24: 1100-1106Crossref PubMed Scopus (46) Google Scholar, 11Napiorkowska M. Boilevin J. Darbre T. Reymond J.L. Locher K.P. Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide.Sci. Rep. 2018; 8: 16297Crossref PubMed Scopus (15) Google Scholar). In animals, plants, and fungi, OST is a multi-subunit protein complex in which the STT3 protein is the conserved catalytic subunit containing the active site. Stt3p is homologous to the single-subunit OST enzymes found in some bacteria (e.g., PglB from C. lari), archaea (e.g., AglB from Pyrococcus furiosus), and eukaryotic kinetoplastids (e.g., STT3 from Leishmania major and Trypanosoma brucei) (12Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47R-62RCrossref PubMed Scopus (402) Google Scholar). Many residues essential for OST function are highly conserved, and the superposition of the substrate-bound structures of PglB to the structure of the yeast Stt3p reveals that the active sites of Stt3 and PglB are very similar (13Wild R. Kowal J. Eyring J. Ngwa E.M. Aebi M. Locher K.P. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.Science. 2018; 359: 545-550Crossref PubMed Scopus (87) Google Scholar, 14Bai L. Wang T. Zhao G. Kovach A. Li H. The atomic structure of a eukaryotic oligosaccharyltransferase complex.Nature. 2018; 555: 328-333Crossref PubMed Scopus (52) Google Scholar). The key catalytic residues that bind the peptide and LLO substrates in the substrate-bound PglB structures are conserved in the yeast Stt3p and located at the same positions with respect to both substrates and the coordinating metal ion (6Lizak C. Gerber S. Numao S. Aebi M. Locher K.P. X-ray structure of a bacterial oligosaccharyltransferase.Nature. 2011; 474: 350-355Crossref PubMed Scopus (258) Google Scholar, 10Napiorkowska M. Boilevin J. Sovdat T. Darbre T. Reymond J.L. Aebi M. Locher K.P. Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase.Nat. Struct. Mol. Biol. 2017; 24: 1100-1106Crossref PubMed Scopus (46) Google Scholar). Despite the same reaction mechanism, major differences in the substrate specificities of OSTs from different organisms are reported. In most eukaryotes, the LLO substrate is large and contains the GlcNAc2Man9Glc3 oligosaccharide, but smaller glycan substrates exist (1Schwarz F. Aebi M. Mechanisms and principles of N-linked protein glycosylation.Curr. Opin. Struct. Biol. 2011; 21: 576-582Crossref PubMed Scopus (438) Google Scholar, 15Samuelson J. Banerjee S. Magnelli P. Cui J. Kelleher D.J. Gilmore R. Robbins P.W. The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1548-1553Crossref PubMed Scopus (206) Google Scholar, 16Kelleher D.J. Banerjee S. Cura A.J. Samuelson J. Gilmore R. Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms.J. Cell Biol. 2007; 177: 29-37Crossref PubMed Scopus (39) Google Scholar). It also appears that the transferred oligosaccharides are shorter in prokaryotic protein glycosylation. In terms of the polypeptide substrate, the N-X-(S/T) sequon requirement remains the same for all OSTs, yet animals, plants, and fungi have a wider range of protein substrates and a larger number of glycosylation sites modified in their proteomes (1Schwarz F. Aebi M. Mechanisms and principles of N-linked protein glycosylation.Curr. Opin. Struct. Biol. 2011; 21: 576-582Crossref PubMed Scopus (438) Google Scholar, 17Zielinska D.F. Gnad F. Wisniewski J.R. Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.Cell. 2010; 141: 897-907Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 18Zielinska D.F. Gnad F. Schropp K. Wisniewski J.R. Mann M. Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core machinery.Mol. Cell. 2012; 46: 542-548Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), compared with bacteria, archaea, and unicellular eukaryotic kinetoplastids (19Scott N.E. Nothaft H. Edwards A.V. Labbate M. Djordjevic S.P. Larsen M.R. Szymanski C.M. Cordwell S.J. Modification of the Campylobacter jejuni N-linked glycan by EptC protein-mediated addition of phosphoethanolamine.J. Biol. Chem. 2012; 287: 29384-29396Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 20Luo Q. Upadhya R. Zhang H. Madrid-Aliste C. Nieves E. Kim K. Angeletti R.H. Weiss L.M. Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry.Microbes Infect. 2011; 13: 1199-1210Crossref PubMed Scopus (28) Google Scholar, 21Atwood 3rd, J.A. Minning T. Ludolf F. Nuccio A. Weatherly D.B. Alvarez-Manilla G. Tarleton R. Orlando R. Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling.J. Proteome Res. 2006; 5: 3376-3384Crossref PubMed Scopus (65) Google Scholar). This increase in glycosylated sites correlates with an increase in OST complexity through the acquisition of additional subunits. It is thought that the additional noncatalytic subunits enhance glycosylation efficiency of the catalytic STT3 protein by facilitating protein and LLO substrate binding (1Schwarz F. Aebi M. Mechanisms and principles of N-linked protein glycosylation.Curr. Opin. Struct. Biol. 2011; 21: 576-582Crossref PubMed Scopus (438) Google Scholar, 12Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47R-62RCrossref PubMed Scopus (402) Google Scholar). The prototypic OST complex from the budding yeast Saccharomyces cerevisiae is composed of the eight subunits Ost1p, Ost2p, Ost4p, Ost5p, Stt3p, Swp1p, Wbp1p, and either Ost3p or Ost6p (12Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47R-62RCrossref PubMed Scopus (402) Google Scholar). Ost3p and Ost6p are nonessential homologous oxidoreductases that assemble as the last subunit into separate OST complexes, resulting in two isoforms of yeast OST that coexist (22Spirig U. Bodmer D. Wacker M. Burda P. Aebi M. The 3.4-kDa Ost4 protein is required for the assembly of two distinct oligosaccharyltransferase complexes in yeast.Glycobiology. 2005; 15: 1396-1406Crossref PubMed Scopus (53) Google Scholar, 23Schwarz M. Knauer R. Lehle L. Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit.FEBS Lett. 2005; 579: 6564-6568Crossref PubMed Scopus (43) Google Scholar, 24Mueller S. Wahlander A. Selevsek N. Otto C. Ngwa E.M. Poljak K. Frey A.D. Aebi M. Gauss R. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly.Mol. Biol. Cell. 2015; 26: 2596-2608Crossref PubMed Scopus (29) Google Scholar). Multicellular animals and plants have an additional layer of complexity in that they express two different OST complexes that contain one of the two Stt3 paralogs, Stt3A and Stt3B (25Kelleher D.J. Karaoglu D. Mandon E.C. Gilmore R. Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties.Mol. Cell. 2003; 12: 101-111Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 26Jiang L. Zhu X. Chen J. Yang D. Zhou C. Hong Z. Two conserved oligosaccharyltransferase catalytic subunits required for N-glycosylation exist in Spartina alterniflora.Bot. Stud. 2015; 56: 31Crossref PubMed Scopus (3) Google Scholar, 27Cherepanova N. Shrimal S. Gilmore R. N-linked glycosylation and homeostasis of the endoplasmic reticulum.Curr. Opin. Cell Biol. 2016; 41: 57-65Crossref PubMed Scopus (99) Google Scholar). The Stt3A complex associates directly with the translocon via the subunit DC2 and performs cotranslational glycosylation, whereas the Stt3B complex incorporates an oxidoreductase subunit instead of DC2 and performs posttranslocational glycosylation (13Wild R. Kowal J. Eyring J. Ngwa E.M. Aebi M. Locher K.P. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.Science. 2018; 359: 545-550Crossref PubMed Scopus (87) Google Scholar, 28Ruiz-Canada C. Kelleher D.J. Gilmore R. Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms.Cell. 2009; 136: 272-283Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 29Shrimal S. Trueman S.F. Gilmore R. Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST.J. Cell Biol. 2013; 201: 81-95Crossref PubMed Scopus (59) Google Scholar, 30Shrimal S. Cherepanova N.A. Gilmore R. DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon.J. Cell Biol. 2017; 216: 3625-3638Crossref PubMed Scopus (27) Google Scholar, 31Ramirez A.S. Kowal J. Locher K.P. Cryo-electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B.Science. 2019; 366: 1372-1375Crossref PubMed Scopus (29) Google Scholar). In the yeast OST, an Stt3B-type OST, the oxidoreductases Ost3p or Ost6p are incorporated, equivalent to the mutually exclusive incorporation of the TUSC3 and MagT1 oxidoreductase subunits in mammalian Stt3B complexes (31Ramirez A.S. Kowal J. Locher K.P. Cryo-electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B.Science. 2019; 366: 1372-1375Crossref PubMed Scopus (29) Google Scholar, 32Cherepanova N.A. Shrimal S. Gilmore R. Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins.J. Cell Biol. 2014; 206: 525-539Crossref PubMed Scopus (64) Google Scholar). Interestingly, while nearly all organisms that express multi-subunit OSTs encode an oxidoreductase subunit (OST3 homolog) (12Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47R-62RCrossref PubMed Scopus (402) Google Scholar), two functional homologs of oxidoreductases appear to be present in all vertebrates and some fungi, suggesting an important function of such redundancy (33Schulz B.L. Aebi M. Analysis of glycosylation site occupancy reveals a role for Ost3p and Ost6p in site-specific N-glycosylation efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Ost3p and Ost6p are both oxidoreductases that can interact with the polypeptide substrate and are thought to slow down the oxidative folding of the glycoprotein substrate to transiently improve accessibility of available glycosylation sequons and increase glycosylation efficiency (34Schulz B.L. Stirnimann C.U. Grimshaw J.P. Brozzo M.S. Fritsch F. Mohorko E. Capitani G. Glockshuber R. Grutter M.G. Aebi M. Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 11061-11066Crossref PubMed Scopus (104) Google Scholar, 35Mohorko E. Owen R.L. Malojcic G. Brozzo M.S. Aebi M. Glockshuber R. Structural basis of substrate specificity of human oligosaccharyl transferase subunit N33/Tusc3 and its role in regulating protein N-glycosylation.Structure. 2014; 22: 590-601Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Indeed, the high-resolution structures of the yeast and the mammalian Stt3B OST show that this subunit not only directly interacts with the catalytic subunit, Stt3p, but its thioredoxin domain is also positioned right across from the Stt3p active site (13Wild R. Kowal J. Eyring J. Ngwa E.M. Aebi M. Locher K.P. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.Science. 2018; 359: 545-550Crossref PubMed Scopus (87) Google Scholar, 14Bai L. Wang T. Zhao G. Kovach A. Li H. The atomic structure of a eukaryotic oligosaccharyltransferase complex.Nature. 2018; 555: 328-333Crossref PubMed Scopus (52) Google Scholar, 31Ramirez A.S. Kowal J. Locher K.P. Cryo-electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B.Science. 2019; 366: 1372-1375Crossref PubMed Scopus (29) Google Scholar). In yeast, Ost3p- and Ost6p-containing complexes have different peptide substrate preferences in vivo, and the Ost3p-containing complex (OST3 complex) is required for efficient glycosylation of a larger subset of proteins than the Ost6p-containing complex (OST6 complex) (34Schulz B.L. Stirnimann C.U. Grimshaw J.P. Brozzo M.S. Fritsch F. Mohorko E. Capitani G. Glockshuber R. Grutter M.G. Aebi M. Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 11061-11066Crossref PubMed Scopus (104) Google Scholar). Furthermore, the OST3 complex is more abundant in yeast (33Schulz B.L. Aebi M. Analysis of glycosylation site occupancy reveals a role for Ost3p and Ost6p in site-specific N-glycosylation efficiency.Mol. Cell Proteomics. 2009; 8: 357-364Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 36Jamaluddin M.F. Bailey U.M. Schulz B.L. Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.Mol. Cell Proteomics. 2014; 13: 3286-3293Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 37Poljak K. Selevsek N. Ngwa E. Grossmann J. Losfeld M.-E. Aebi M. Quantitative profiling of N-linked glycosylation machinery in yeast Saccharomyces cerevisiae.Mol. Cell Proteomics. 2018; 17: 18-30Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) and has a higher relative enzymatic activity than the OST6 complex in vitro (38Harada Y. Buser R. Ngwa E.M. Hirayama H. Aebi M. Suzuki T. Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.J. Biol. Chem. 2013; 288: 32673-32684Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 39Yamasaki T. Kohda D. Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase.J. Biol. Chem. 2020; 295: 16072-16085Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). To understand the differences between the OST3 and OST6 complexes, we characterized the substrate specificities and enzyme kinetics of the two OST isoforms in vitro. We found that the two complexes had similar affinities to short, synthetic peptide and LLO substrates but differed significantly in their catalytic efficiency. The two isoforms of the yeast OST complex that contain either of the functional homologs, Ost3p or Ost6p, were purified separately from strains expressing only one type of complex by deleting either OST6 or OST3 and overexpressing the desired subunit (OST3 or OST6, respectively) to ensure the assembly of complete complexes in the cell (22Spirig U. Bodmer D. Wacker M. Burda P. Aebi M. The 3.4-kDa Ost4 protein is required for the assembly of two distinct oligosaccharyltransferase complexes in yeast.Glycobiology. 2005; 15: 1396-1406Crossref PubMed Scopus (53) Google Scholar, 23Schwarz M. Knauer R. Lehle L. Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit.FEBS Lett. 2005; 579: 6564-6568Crossref PubMed Scopus (43) Google Scholar). Insertion of a 1D4 epitope tag at the C-terminus of the Ost4p subunit in these strain backgrounds allowed for a very efficient purification of complete OST complexes of a single isoform, as shown previously for determining the structure of the yeast OST (13Wild R. Kowal J. Eyring J. Ngwa E.M. Aebi M. Locher K.P. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.Science. 2018; 359: 545-550Crossref PubMed Scopus (87) Google Scholar). Silver staining of purified OST complexes showed that all eight subunits were present and either Ost3p or Ost6p was solely incorporated into each respective complex (Fig. 1A). Both complexes also ran as single monodispersed complexes in size-exclusion chromatography and had a very similar size (Fig. 1B). Aside from the presence of either Ost3p or Ost6p, the only other difference observed between the purified OST isoforms was that the OST6 complex contained hypoglycosylated subunits (Fig. 1A). Analysis of the glycosylation site occupancy of the OST complex glycoproteins Stt3p, Wbp1p, and Ost1p by mass spectrometry confirmed that the OST6 complex was generally less efficiently glycosylated than the OST3 complex, but the Stt3p N539 glycosite was fully glycosylated in both OST complexes (Fig. 1C). Both Wbp1p glycosylation sites were fully modified in the OST3 complex, but their site occupancy was reduced in the OST6 complex, particularly that of Wbp1p N60 (reduced by 50%). Regarding the Ost1p glycosylation sites, only Ost1p N99 was fully glycosylated in the OST3 complex and the occupancies of Ost1p N99 and N217 were only slightly reduced in the OST6 complex, but not to the same extent as that observed for the Wbp1p glycosites. The glycan structures at each glycosylation site were determined by mass spectrometry. We found glycan heterogeneity at each site analyzed, but the site-specific glycan structure profile was very similar in both OST3 and OST6 complexes (Fig. 1D). In agreement with our understanding of the yeast glycan modification pathway in the ER and Golgi (40Xu C. Ng D.T. Glycosylation-directed quality control of protein folding.Nat. Rev. Mol. Cell Biol. 2015; 16: 742-752Crossref PubMed Scopus (199) Google Scholar), the most abundant glycan structure on Stt3p N539, Wbp1p N60, and Ost1p N217 was identified as Man8GlcNAc2, confirming a previous report on Stt3p N539 (41Li G. Yan Q. Nita-Lazar A. Haltiwanger R.S. Lennarz W.J. Studies on the N-glycosylation of the subunits of oligosaccharyl transferase in Saccharomyces cerevisiae.J. Biol. Chem. 2005; 280: 1864-1871Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The glycan profile of Wbp1p N332 showed that it carried either Man8GlcNAc2 or Man7GlcNAc2 structures. Ost1p N99 carried either Man8GlcNAc2 or Man9GlcNAc2 structures, but we also detected some larger glycans with up to ten hexose units (Fig. 1D). OST activity was determined using a short peptide labeled with the TAMRA (tetramethylrhodamine) fluorophore and synthetic LLO analogs with two GlcNAc residues (chitobiose) as the sugar moiety, as used previously (13Wild R. Kowal J. Eyring J. Ngwa E.M. Aebi M. Locher K.P. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.Science. 2018; 359: 545-550Crossref PubMed Scopus (87) Google Scholar, 42Ramirez A.S. Boilevin J. Biswas R. Gan B.H. Janser D. Aebi M. Darbre T. Reymond J.L. Locher K.P. Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs.Glycobiology. 2017; 27: 525-535Crossref PubMed Scopus (22) Google Scholar). Incubation of these synthetic substrates with purified yeast OST resulted in the formation of a fluorophore-labeled glycopeptide product (Fig. 2A). We detected and quantified the formation of glycopeptide product using reverse phase UPLC (ultra performance liquid chromatography) (43Naegeli A. Michaud G. Schubert M. Lin C.W. Lizak C. Darbre T. Reymond J.L. Aebi M. Substrate specificity of cytoplasmic N-glycosyltransferase.J. Biol. Chem. 2014; 289: 24521-24532Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) instead of a gel-based visualization of glycopeptide formation (Fig. 2B). The peak corresponding to the earlier-eluting glycopeptide increased with reaction time, and peak integration allowed an accurate and sensitive quantification of product formation. A quantitative analysis of the peptide substrate specificity of the OST3 complex in vitro was first performed using the fluorescently labeled hexapeptide TAMRA-DANYTK-NH2 as the acceptor peptide (44Schwarz F. Fan Y.Y. Schubert M. Aebi M. Cytoplasmic N-glycosyltransferase of Actinobacillus pleuropneumoniae is an inverting enzyme and recognizes the NX(S/T) consensus sequence.J. Biol. Chem. 2011; 286: 35267-35274Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The initial reaction velocity was measured at various peptide concentrations and a fixed synthetic LLO concentration (Fig. 2C). The data were fitted according to Michaelis–Menten kinetics and apparent KM and kcat values were derived (Table 1). Saturation of the peptide substrate could not be reached, but the high apparent KM of around 200 μM indicated that the short peptide used was a rather poor substrate. To obtain a more optimal peptide substrate for the yeast OST, we screened a library of peptide sequences containing a glycosylation sequon derived from a list of efficiently glycosylated yeast proteins in vivo (37Poljak K. Selevsek N. Ngwa E. Grossmann J. Losfeld M.-E. Aebi M. Quantitative profiling of N-linked glycosylation machinery in yeast Saccharomyces cerevisiae.Mol. Cell Proteomics. 2018; 17: 18-30Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) (Table S1). The screen was performed by adding unlabeled synthetic peptides as a competitor substrate to in vitro reactions containing the fluorescently labeled TAMRA-DANYTK. Some peptides slightly inhibited the turnover of the labeled TAMRA-DANYTK peptide, while Peptide 5 (ADTYANATSDVL) inhibited the reaction by 50% (Fig. 2D). To directly show that this peptide sequence was indeed a better acceptor substrate than TAMRA-DANYTK, we measured the initial turnover of TAMRA-labeled Peptide 5 (TAMRA-ADTYANATSDVL) and a shorter version of Peptide 5 of only six amino acid residues (TAMRA-YANATS). Fluorophore-labeled Peptide 5 was glycosylated 1.5 times faster than TAMRA-DANYTK and the short version was glycosylated 14 times faster (Table S2). Direct quantitative analysis using the TAMRA-YANATS peptide as the substrate (Fig. 2E) revealed a 3.5-fold lower apparent KM and a 3.5-fold higher maximal initial turnover rate (Table 1): TAMRA-YANATS was over ten times more specific to the yeast OST3 complex than TAMRA-DANYTK. For further experiments, the TAMRA-YANATS peptide substrate was used.Table 1Kinetic parameters for peptide and LLO substrates with OST3 and OST6 complexesPeptide parametersKM (μM)kcat (min−1)kcat/KM (min-1 μM−s1)OST3 complexTAMRA-DANYTK201.0 ± 51.33.9 ± 0.60.02TAMRA-YANATS55.6 ± 6.514.5 ± 0.70.26OST6 complexTAMRA-YANATS44.8 ± 5.72.3 ± 0.20.05Synthetic LLO (GlcNAc2-PP-Lipid) parametersLipid nameLipid lengthOST3 complexCitronellylC10n.d.n.d.n.d.FarnesylC1567.9 ± 6.71.7 ± 0.10.02CitronellylnerylC2020.6 ± 0.69.9 ± 0.060.48CitronellylfarnesylC2512.5 ± 2.34.5 ± 0.30.36OST6 complexCitronellylnerylC2025.5 ± 1.71.9 ± 0.010.07TAMRA, tetramethylrhodamine.n.d. = activity not detected with TAMRA-DANYTK acceptor substrate.The apparent KM and kcat values were determined from data shown in Figures 2, C and E, 3B, and 5, C and D. Errors represent standard deviations from the mean of three replicates (n = 3) fitted by nonlinear regression using the Michaelis–Menten equation in Prism. Open table in a new tab TAMRA, tetramethylrhodamine. n.d. = activity not detected with TAMRA-DANYTK acceptor substrate. The apparent KM and kcat values were determined from data shown in Figures 2, C and E, 3B, and 5, C and D. Errors represent standard deviations from the mean of three replicates (n = 3) fitted by nonlinear regression using the Michaelis–
Referência(s)