Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms

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