Carbohydrate Composition Analysis of Glycoconjugates By Gas-Liquid Chromatography. Mass Spectrometry. Metabolic Radiolabeling of Glycoconjugates. Nonmetabolic Radiolabeling and Tagging of Glyconconjugates. Enzymatic Deglycosylation of Asparagine-Linked Glycans: Purification, Properties and Specificity of Oligosaccharide-Cleaving Enzymes from Flavobacterium Meningosepticum. Release of Oligosaccharides from Glycoproteins By Hydrazinolysis. Use of Lectins in Analysis of Clycoconjugates. Saccharide Linkage Analysis Using Methylation and Other Techniques. Mass Spectrometry of Carbohydrates-Containing Bipolymers. 1H Nuclear Magnetic Resonance Spectroscopy of Carbohydrate Chains of Glycoproteins. Determination of Sialic Acids. Size Fractionation of Oligosaccharides. High-Ph Anion-Exchange Chromatography of Glycoprotein-Derived Carbohydrates. High-Performance Liquid Chromatography of Pyridylaminated Saccharides. High-Performance Liquid Chromatography of Oligosaccharides. High-Resolution Polycrylamide Gel Electro-Phoresis of Oligosaccharides. Glycosidases in Structural Analysis. Glycosyltransferases in Glycosidase Inhibitors in Study of Glycoconjugates. Synthesis and Use of Azido-Substituted Nucleoside Diphosphate Sugar Photoaffinity Analogs. Glycoform Analysis of Glycoproteins. Isolation of Glycosphingolipids. Thin-Layer Chromatography of Glycosphingolids. Isolation and Characterization of Proteoglycans. Structural Analysis of Glycosylphosphatidylinositol Anchors. Detection of O-Linked N-Acetylglucosamine (O-Glcnac) on Cytoplasmic and Nuclear Proteins. Identification of Polysialic Acids in Glycoconjugates. Neoglycolipds: Probes of Oligosaccharide Structure, Antigenicity, And Function. Author Index. Subject Index.
Membrane fusion events are required in three steps in sea urchin fertilization: the acrosome reaction in sperm, fusion of the plasma membrane of acrosome-reacted sperm with the plasma membrane of the egg, and exocytosis of the contents of the egg cortical granules. We recently reported the involvement of a Zn2+-dependent metalloendoprotease in the acrosome reaction (Farach, H. C., D. I. Mundy, W. J. Strittmatter, and W. J. Lennarz. 1987. J. Biol. Chem. 262:5483-5487). In the current study, we investigated the possible involvement of metalloendoproteases in the two other fusion events of fertilization. The use of inhibitors of metalloendoproteases provided evidence that at least one of the fusion events subsequent to the acrosome reaction requires such enzymes. These inhibitors did not block the binding of sperm to egg or the process of cortical granule exocytosis. However, sperm-egg fusion, assayed by the ability of the bound sperm to establish cytoplasmic continuity with the egg, was inhibited by metalloendoprotease substrate. Thus, in addition to the acrosome reaction, an event in the gamete fusion process requires a metalloendoprotease.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAromatic boronic acids. Synthesis of o-boronophenylalanineJames R. Kuszewski, William J. Lennarz, and Harold R. SnyderCite this: J. Org. Chem. 1968, 33, 12, 4479–4483Publication Date (Print):December 1, 1968Publication History Published online1 May 2002Published inissue 1 December 1968https://pubs.acs.org/doi/10.1021/jo01276a039https://doi.org/10.1021/jo01276a039research-articleACS PublicationsRequest reuse permissionsArticle Views233Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Small quantities of highly purified granulocytic pyrogen have been separated from contaminating proteins by disc electrophoresis in polyacrylamide gel. The biologically active material thus isolated was shown to be electrophoretically homogeneous at pH 9 and pH 3.8. Earlier work on the chemical properties of the pyrogen molecule has been extended to include: (a) estimation of its molecular weight by gel filtration; (b) demonstration of free sulfhydryl groups essential for its biological activity; and (c) evidence that it is not inactivated by exhaustive extraction with ethanolether or n-heptane.
Retroviral infection is associated with immunosuppression, which has been shown to be due, in part, to the action of the envelope protein p15E. We studied a synthetic peptide (CKS-17) homologous to a highly conserved domain of the retroviral envelope protein p15E, which, when conjugated to BSA (CKS-17-BSA), can inhibit IL-1- and phorbol ester-mediated responses in cultured murine thymoma cells, and Ca2(+)- and phosphatidylserine-dependent protein kinase C (PKC) activity of cell homogenates. We characterized the mechanism of inhibition of PKC by the peptide. Using PKC purified from rat brain we found that CKS-17-BSA inhibited PKC-catalyzed Ca2(+)- and phosphatidylserine-dependent histone phosphorylation with an estimated ID50 of 4 microM. CKS-17-BSA did not inhibit the catalytic subunit of cAMP-dependent protein kinase. CKS-17-BSA also inhibited the Ca2(+)- and PS-independent activity of a catalytic fragment of PKC that was generated by limited trypsin treatment. However, CKS-17-BSA did not act as a competitive inhibitor of PKC with respect to ATP or phosphoacceptor substrate, despite the similarity between the CKS-17 sequence and substrates and pseudosubstrates of PKC. We conclude that this peptide homologue of a retroviral envelope protein has a novel mechanism of inhibition of PKC.
In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT) is composed of nine different transmembrane proteins. Using a glycosylatable peptide containing a photoprobe, we previously found that only one essential subunit, Ost1p, was specifically labeled by the photoprobe and recently have shown that it does not contain the recognition domain for the glycosylatable sequence Asn-Xaa-Thr/Ser. In this study we utilized additional glycosylatable peptides containing two photoreactive groups and found that these were linked to Stt3p and Ost3p. Stt3p is the most conserved subunit in the OT complex, and therefore 21 block mutants in the lumenal region were prepared. Of the 14 lethal mutant proteins only two, as well as one temperature-sensitive mutant protein, were incorporated into the OT complex. However, using microsomes prepared from these three strains, the labeling of Ost1p was markedly decreased upon photoactivation with the Asn-Bpa-Thr photoprobe. Based on the block mutants single amino acid mutations were prepared and analyzed. From all of these results, we conclude that the sequence from residues 516 to 520, WWDYG in Stt3p, plays a central role in glycosylatable peptide recognition and/or the catalytic glycosylation process.
In an effort to identify the polypeptide chain of glucosylphosphodolichol synthase (EC 2.4.1.117), yeast microsomal membranes were allowed to react with 5-azido[beta-32P]UDPGlc, a photoactive analogue of UDPGlc, which is a substrate for this enzyme. Upon photolysis the 32P-labeled probe was shown to link covalently to a 35-kDa protein present in microsomal membranes prepared from several wild-type yeast strains. Binding was either reduced or absent in the microsomal membranes from two yeast mutants (alg5 and dpg1) that are known to be defective in the synthesis of glucosylphosphodolichol. The microsomes isolated from a heterozygous diploid strain alg5::dpg1 generated from these two mutants exhibited partial restoration of both the ability to photolabel the 35-kDa protein and the ability to catalyze the synthesis of glucosylphosphodolichol. Microsomal membranes from a mutant strain that synthesized glucosylphosphodolichol but lacked the ability to transfer the glucosyl residue to the growing lipid-linked oligosaccharide (alg6) exhibited labeling with 5-azido[beta-32P]UDPGlc comparable to that found in microsomes from the wild-type strain. In all cases photoinsertion of the probe into the 35-kDa protein correlated with the level of synthase assayed in the microsomal membranes. These results strongly support the conclusion that the 35-kDa protein labeled in these experiments is a component of glucosylphosphodolichol synthase.
In Saccharomyces cerevisiae, oligosaccharyl transferase (OT) consists of nine different subunits. Three of the essential gene products, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins. To study the function of the N-glycosylation of these proteins, we prepared single or multiple N-glycosylation site mutations in each of them. We established that the four potential N-glycosylation sites in Ost1p and the two potential N-glycosylation sites in Wbp1p were occupied in the mature proteins. Interestingly, none of the N-glycosylation sites in these two proteins was conserved, and no defect in growth or OT activity was observed when the N-glycosylation sites were mutated to block N-glycosylation in either subunit. However, in the third glycosylated subunit, Stt3p, there are two adjacent potential N-glycosylation sites (N535NTWN539NT) that, in contrast to the other subunits, are highly conserved in eukaryotic organisms. Mass spectrometric analysis of a tryptic digest of Stt3p showed that the peptide containing the two adjacent N-glycosylation sites was N-glycosylated at one site. Furthermore, the glycan chain identified as Man8GlcNAc2 is found linked only to Asn539. Mutation experiments were carried out at these two sites. Four single amino acid mutations blocking either N-glycosylation site (N535Q, T537A, N539Q, and T541A) resulted in strains that were either lethal or extremely temperature sensitive. However, other mutations in the two N-glycosylation sites N535NTWN539NT (N536Q, T537S, N540Q, and T541S), did not exhibit growth defects. Based on these studies, we conclude that N-glycosylation of Stt3p at Asn539 is essential for its function in the OT complex. In Saccharomyces cerevisiae, oligosaccharyl transferase (OT) consists of nine different subunits. Three of the essential gene products, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins. To study the function of the N-glycosylation of these proteins, we prepared single or multiple N-glycosylation site mutations in each of them. We established that the four potential N-glycosylation sites in Ost1p and the two potential N-glycosylation sites in Wbp1p were occupied in the mature proteins. Interestingly, none of the N-glycosylation sites in these two proteins was conserved, and no defect in growth or OT activity was observed when the N-glycosylation sites were mutated to block N-glycosylation in either subunit. However, in the third glycosylated subunit, Stt3p, there are two adjacent potential N-glycosylation sites (N535NTWN539NT) that, in contrast to the other subunits, are highly conserved in eukaryotic organisms. Mass spectrometric analysis of a tryptic digest of Stt3p showed that the peptide containing the two adjacent N-glycosylation sites was N-glycosylated at one site. Furthermore, the glycan chain identified as Man8GlcNAc2 is found linked only to Asn539. Mutation experiments were carried out at these two sites. Four single amino acid mutations blocking either N-glycosylation site (N535Q, T537A, N539Q, and T541A) resulted in strains that were either lethal or extremely temperature sensitive. However, other mutations in the two N-glycosylation sites N535NTWN539NT (N536Q, T537S, N540Q, and T541S), did not exhibit growth defects. Based on these studies, we conclude that N-glycosylation of Stt3p at Asn539 is essential for its function in the OT complex. Oligosaccharyl transferase (OT) 1The abbreviations used are: OT, oligosaccharyl transferase; 5-FOA, 5-fluoroorotic acid; HA, hemagglutinin; Endo H, endo-β-N-acetylglucosaminidase H; MS/MS, tandem mass spectrometry; ER, endoplasmic reticulum; PNGase F, peptide N-glycosidase F; MS/MS/MS, MS/MS spectra subjected to the next round of MS. catalyzes the N-glycosylation of most secretory and membrane-bound proteins. In Saccharomyces cerevisiae, OT consists of nine different subunits, of which five are essential gene products (1Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar). At present, the detailed structural organization of the OT complex, as well as the function of each of the OT subunits, is unclear. Growing evidence supports the proposal that one of the essential OT subunits, Stt3p, is involved directly in the substrate recognition process (2Yan Q. Lennarz W.J. J. Biol. Chem. 2002; 277: 47692-47700Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 3Wacker M. Linton D. Hitchen P.G. Nita-Lazar M. Haslam S.M. North S.J. Panico M. Morris H.R. Dell A. Wren B.W. Aebi M. Science. 2002; 298: 1790-1793Crossref PubMed Scopus (628) Google Scholar, 4Nilsson I. Kelleher D.J. Miao Y. Shao Y. Kreibich G. von Gilmore R. Heijne G. Johnson A.E. J. Cell Biol. 2003; 161: 715-725Crossref PubMed Scopus (120) Google Scholar). In addition, Stt3p has been shown to be involved in cell wall β-1,6-glycan biosynthesis in S. cerevisiae and to interact with protein kinase cascade components via its N-terminal domain, which is oriented toward the cytosol. This function may be independent of its role in the recognition process of substrates, which involves the C-terminal domain that is oriented toward the lumen (5Chavan M. Suzuki T. Rekowicz M. Lennarz W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15381-15386Crossref PubMed Scopus (28) Google Scholar, 6Chavan M. Rekowicz M. Lennarz W. J. Biol. Chem. 2003; 278: 51441-51447Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The nine subunits of yeast OT have been hypothesized to comprise three subcomplexes, Ost1p-Ost5p, Ost2p-Swp1p-Wbp1p, and Stt3p-Ost4p-Ost3p (1Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar), although recent data are not completely consistent with this model (7Yan A. Ahmed E. Yan Q. Lennarz W.J. J. Biol. Chem. 2003; 278: 33078-33087Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). It is of interest that each of these three subcomplexes contains one of the three glycoproteins, Ost1p, Wbp1p, or Stt3p (1Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar). The importance of the N-glycosylation of OT subunits with respect to their function is unknown. In the current study, we have addressed this question by site-directed mutation of potential N-glycosylation sites on the Ost1p, Wbp1p, and Stt3p subunits. In addition, because our knowledge of the minimal spacing required for efficient N-glycosylation at two adjacent sites was limited, we also addressed this question in the current study. Strains and Plasmids—The haploid strains QYY500 (MAT a ade2 can1 his3 leu2 trp1 ura3 Δost1::his5+ (Schizosaccharomyces pombe) pRS316-OST1), L2 (MAT a ade2 can1 his3 leu2 trp1 ura3 Δwbp1::his5+ (S. pombe) pRS316-WBP1), and QYY700 (MAT a ade2 can1 his3 leu2 trp1 ura3 Δstt3::his5+ (S. pombe) YEP352-STT3) were used in the Ost1p, Wbp1p, and Stt3p mutation assays, respectively. Plasmids pRS314-OST1HA, pRS314-WBP1HA and pRS314-STT3HA were constructed as described previously (2Yan Q. Lennarz W.J. J. Biol. Chem. 2002; 277: 47692-47700Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 8Yan Q. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15994-15999Crossref PubMed Scopus (17) Google Scholar, 9Li G. Yan Q. Oen H.O. Lennarz W.J. Biochemistry. 2003; 42: 11032-11039Crossref PubMed Scopus (12) Google Scholar). The haploid strain AYY7 (7Yan A. Ahmed E. Yan Q. Lennarz W.J. J. Biol. Chem. 2003; 278: 33078-33087Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) was used to check the N-glycosylation of Ost6p. PCR Mutagenesis for Block and Point Mutants—PCR mutagenesis was performed using a site-directed mutagenesis kit following the manufacturer's protocol (Stratagene). For all of the block and point mutations mentioned in this study, pRS314-OST1HA, pRS314-WBP1HA, and pRS314-STT3HA were used as the PCR templates for Ost1p, Wbp1p, and Stt3p mutagenesis, respectively. Mutagenized plasmids were sequenced, and those with the expected sequence were transformed into QYY500, L2, and QYY700. The transformants were selected for Trp and Ura prototrophy and further selected on plates containing 5-fluoroorotic acid (5-FOA) (Sigma) for growth. Spotting Assay for Growth—To determine the growth difference between yeast transformants carrying either Ost1p or Wbp1p mutations and wild type control, an equal number of cells were collected after the strains grew to early log phase in –Trp medium at 25 °C. Then 7 μl of 1:10 serial dilutions of the cells were spotted onto –Trp plates, incubated at 25, 30, or 37 °C for 2 days, and examined for growth. For Stt3p wild type or mutants, cells were grown initially in the –Trp–Ura medium and then spotted on –Trp–Ura and –Trp+FOA plates. Determination of OT Activity—The oligosaccharyl transferase activity was carried out as described previously (9Li G. Yan Q. Oen H.O. Lennarz W.J. Biochemistry. 2003; 42: 11032-11039Crossref PubMed Scopus (12) Google Scholar). The glycosylation activity was expressed as the amount of labeled glycopeptide formed (in cpm)/unit of protein/unit of time. Immunoprecipitation and Co-immunoprecipitation—QYY700 carrying wild type Stt3pHA or mutants (N60Q, N535Q, T537A, N539Q, T541A, and T537A/N539Q) was used for co-immunoprecipitation. Co-immunoprecipitation under a mild detergent condition was carried out as described previously (9Li G. Yan Q. Oen H.O. Lennarz W.J. Biochemistry. 2003; 42: 11032-11039Crossref PubMed Scopus (12) Google Scholar). Immunoprecipitation of Stt3HAp and mutants was performed in harsh detergent buffer. The spheroplasts were suspended in 1% Triton X-100, 0.2% SDS, 150 mm NaCl, 10 mm Hepes (pH 7.5), 5 mm MgCl2, and 1 mm phenylmethylsulfonyl fluoride. The mixture was centrifuged for 20 min at 55,000 × g, and the clarified supernatant was used for immunoprecipitation using monoclonal anti-HA antibody (Covance). Recombinant protein G-agarose beads (Invitrogen) were added to recover the antibody. Samples were eluted from the agarose beads using 0.1 m glycine (pH 3.0) or SDS sample buffer. Endo H and PNGase F Digestion—Endo H and PNGase F digestion was performed according to the manufacturer's protocol (New England Biolabs). Cell lysates of the strains with epitope-tagged Ost1p and Wbp1p were used directly for Endo H and PNGase F digestion. For Stt3HAp wild type and mutants, immunoprecipitates were eluted from agarose beads using 0.1 m glycine (pH 3.0) and then digested with Endo H. Mass Spectrometry—Microsomes from 2000 A660 units of Stt3HAp wild type cells were solubilized and immunoprecipitated as described above. To confirm the identity of the purified protein and to obtain evidence for its glycosylation, analysis by SDS-PAGE and peptide mapping was performed. The protein on the beads was reduced in the SDS sample buffer using Tris(2-carboxyethyl)phosphine hydrochloride (Pierce) and was then carboxyamidomethylated prior to loading onto a SDS-polyacrylamide gel. The band corresponding to Stt3HAp was identified by zinc acetate staining (Bio-Rad) gel, cut out, and digested with trypsin (Promega), essentially as described previously (10Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar). The resulting peptides were desalted on a C18 ZipTip microcolumn (Millipore). The peptides were then fractionated by reversed phase liquid chromatography electrospray ionization mass spectrometry (MS) using a Zorbax C18 capillary column (0.3 mm diameter) (Agilent) with a 60 min/linear gradient from 0 to 95% Buffer B (in which Buffer A consisted of 0.1% formic acid, and Buffer B was 95% acetonitrile in 0.1% formic acid) at 5 μl/min. The column effluent was sprayed into an XCT (Agilent) ion trap mass spectrometer. MS/MS of the two major ions in each spectrum was performed automatically to identify peptides, and MS/MS/MS was performed on the glycosylated peptide to confirm its sequence and determine the site of the modification. The data were analyzed using data analysis software (Agilent) combined with online Mascot and Global Protein Machine data base searching programs. Potential Glycosylation Sites in Ost1p, Wbp1p, and Stt3p—In the OT enzyme complex of S. cerevisiae, three of the nine subunits have been reported to be N-linked glycoproteins: Ost1p, Wbp1p, and Stt3p (1Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar). Based on the PROSITE program, four subunits had potential N-glycosylation sites: Ost1p, Wbp1p, Ost6p, and Stt3p (Fig. 1). Among them, Ost1p had six predicted N-glycosylation sites (N18VS, N99ST, N217ET, N336FT, N400NS, and N473VT). However, the first predicted site (N18VS) was located in the signal peptide region (1–22 amino acid), and the last site (N473VT) was located in the cytosolic tail where N-glycosylation is not likely to occur. Wbp1p has two predicted N-glycosylation sites (N60ST and N332DS) that are located in the ER luminal domain and have the potential to be N-glycosylated. Ost6p has only one N-glycosylation site (N175IS). Based on the TMHMM (a method for predicting transmembrane helices based on a hidden Markov model) program, this site is located in the ER lumen and is a potential N-glycosylation site. Stt3p has three predicted N-glycosylation sites (N60NS, N535NT, and N539NT). All of them are located in the ER lumen and clearly are candidates for N-glycosylation. 2H. Kim, personal communication. Interestingly, only one amino acid, Trp538, separates the second and third prospective N-glycosylation sites in Stt3p. To determine whether the N-glycosylation sites in these subunits were conserved, alignment of the homologues of these proteins across eukaryotic species was carried out. In a confirmation of earlier studies (1Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar, 11Silberstein S. Gilmore R. FASEB J. 1996; 10: 849-858Crossref PubMed Scopus (207) Google Scholar), we found that none of the N-glycosylation sites in Ost1p, Wbp1p, and Ost6p was conserved. In contrast, among the three predicted N-glycosylation sites of Stt3p, the second and third N-glycosylation sites (N535NT and N539NT) as well as the tryptophan residue separating them are highly conserved across eukaryotic species, ranging from mammals to yeast (Fig. 2). As also shown in Fig. 2, the sequences flanking the N-glycosylation sites are identical or similar across the eukaryotic species. This is also true of the highly conserved predicted catalytic or glycosylation site recognition domain of Stt3p (W516WDYG). Growth Phenotype and Analysis of N-Glycosylation Site Occupancy—To test the importance of N-glycosylation for the functionality of these proteins, we prepared single amino acid mutations in each, changing the Asn to the similar amino acid Gln at each N-glycosylation site (because OST6 is not an essential gene, we did not mutate its N-glycosylation site). The site and results with all the mutants are summarized in Table I. In Ost1p, in addition to the single amino acid mutations (N99Q, N217Q, N336Q, and N400Q), we prepared a double mutant (N99Q/N217Q) in which the first two N-glycosylation sites were mutated, and we also prepared a strain (N/Q) that was mutated at all four sites. In Wbp1p, in addition to single mutants N60Q and N332Q, a mutant was prepared in which both N-glycosylation sites were mutated (N/Q). In Stt3p, the Asn residues were mutated to Gln (N60Q, N535Q, and N539Q), and the Thr residues in the +2 position relative to Asn were also mutated to Ala at the adjacent N-glycosylation sites (T537A and T541A). A double mutation (T537A/N539Q) was also prepared to block both of the adjacent N-glycosylation sites of Stt3p.Table IGrowth phenotype of the glycosylation sites mutation on S. cerevisiae OT subunitsProteinMutantsGrowth phenotypeGlycosylation change detectedOst1pN99QNormalYesN217QNormalYesN336QNormalYesN400QNormalYesN99Q/N217QNormalYesN/QNormalYesWbp1pN60QNormalYesN332QNormalYesN/QNormalYesStt3pN60QNormalNoN535QLethalYesT537ALethalYesN539QLethalYesT541AExtremely t.s.aTemperature-sensitive.YesT537A/N539QLethalYesN536QNormalNoT537SNormalNoN540QNormalNoT541SNormalNoa Temperature-sensitive. Open table in a new tab First, we tested the growth phenotype of all the mutants. Cells carrying the mutant plasmid (which contains the Trp gene) and wild type gene plasmid (which contains the Ura gene) were patched on –Trp–Ura medium plates and grown for 2 days at 25 °C and then replicated to –Trp+FOA plates to deplete the wild type gene. After incubation at 25 or 37 °C for 2 days, respectively, cell growth was assessed. As summarized in Table I, we found that all the strains bearing the ost1p, wbp1p mutants and the N60Q mutant of Stt3p grew at both temperatures. In further spotting assays, we did not detect any difference in growth between these mutants and the wild type strain (Fig. 3A). In contrast, mutations at the adjacent N-glycosylation sites, N535NT and N539NT of Stt3p, displayed a significant defect; cells carrying mutations N535Q, T537A, and N539Q did not grow at any temperature tested. Cells carrying mutation T541A displayed a severe temperature sensitivity; they barely grew at 25 °C and did not grow at all at 37 °C. This result was confirmed by further spotting assays (Fig. 3B). Next, the effect of these mutations on N-linked glycosylation of the individual subunits was determined by either Endo H or PNGase F digestion followed by Western blot analysis. The yeast cells carrying the ost1p, wbp1p mutants or wild type gene were harvested and lysed, and Endo H or PNGase F digestion was carried out with the wild type proteins (Fig. 4, A and B). The cell lysates with and without digestion were analyzed on a SDS-polyacrylamide gel and blotted with monoclonal anti-HA antibody. As shown in Fig 4A, the wild type Ost1p and all four point mutants exhibit two bands. The double mutant of Ost1p also has two bands and shows lower molecular mass than the highest band of the four single mutants. The four-site mutant (N/Q) of Ost1p migrates the fastest. Similar results are observed with Wbp1p (Fig. 4B) in which the wild type Wbp1p exhibits three bands, and the single mutants (N60Q and N332Q) exhibit lower molecular mass than the wild type. The double mutant has one band almost at the same molecular weight as wild type Wbp1p, after Endo H digestion. Based on these results, we conclude that the four N-glycosylation sites of Ost1p (N99ST, N217ET, N336FT, and N400NS) and the two N-glycosylation sites of Wbp1p (N60ST and N332DS) are occupied in mature wild type proteins (see Fig. 1), but not all the sites have the same level of N-glycosylation. To determine whether Ost6p was N-glycosylated, triple HA tag was integrated in the C terminus of the OST6 gene in the chromosome. Cell lysates treated with Endo H revealed no change in mobility on SDS-PAGE, indicating the absence of N-glycosylation (data not shown). We could not readily detect the protein Stt3HAp using cell lysates for the Western blot analysis. Therefore, we prepared spheroplasts, lysed them in 1% Triton X-100, 0.2% SDS, and immunoprecipitated them with anti-HA monoclonal antibody. The precipitates were analyzed on SDS-PAGE, and following transfer membranes were blotted with anti-HA polyclonal antibody. Stt3HAp always displays a very broad band, but this apparently is not the result of differential N-glycosylation, because the bands are still diffuse even after the removal of the N-glycans by Endo H treatment (Fig. 4C). By comparing the molecular mass of the mutants with or without Endo H digestion, we found that the wild type and all of the single mutants were glycosylated. However, the double mutant that was blocked at both of the adjacent N-glycosylation sites of Stt3p did not exhibit any shift. These results indicate that the first N-glycosylation site (N60NS) is not utilized, and when either the N535NT or N539NT is mutated, the other site is glycosylated. This conclusion was confirmed by further block mutations (Fig. 4D) in which no N-glycosylation occurred in block mutant 537AAAA540, which blocks both of the adjacent sites; however, N-glycosylation occurred in block mutant 541AAA543, which blocks the N-glycosylation site N539NT. This result strongly supports the conclusion that when N539NT is mutated, N535NT becomes glycosylated. Oligosaccharyl Transferase Activity of the OT Subunit Mutants—Two assay methods were used to assess the OT activity of the mutants. One method was to check the N-glycosylation pattern of carboxypeptidase Y in different mutants in vivo. We found that all of the ost1p, wbp1p mutants and the first N-glycosylation site mutant of stt3p (N60Q) failed to exhibit any N-linked glycosylation defect. However, the stt3p mutant T541A displayed a significant underglycosylation pattern in carboxypeptidase Y (Fig. 5A). The in vitro assay using a synthetic peptide as the OT substrate confirmed the in vivo result, that only mutant T541A shows significantly impaired N-glycosylation activity, by a value of 20% compared with wild type (Fig. 5B). What Causes the Growth Phenotype of the Stt3p Glycosylation Site Mutants?—Previously, we found that some OT subunits with certain mutations failed to be incorporated into the OT complex and that these mutations were lethal (9Li G. Yan Q. Oen H.O. Lennarz W.J. Biochemistry. 2003; 42: 11032-11039Crossref PubMed Scopus (12) Google Scholar). To determine whether the lethality of the stt3p mutants was caused by its failure to be incorporated into the OT complex or its functional importance, co-immunoprecipitation under a mild detergent condition was performed. As shown in Fig. 6, all of the mutants are incorporated into the OT complex. Based on this observation we conclude that the N-glycosylation is not required for Stt3p to be incorporated into the OT complex. All the growth defects and the defects in N-glycosylation are observed only with the stt3p mutations in the region that is conserved among eukaryotic species. Obviously, the mutations that were prepared not only blocked the N-glycosylation but changed the amino acid sequence. How can we distinguish the cause of the phenotype? Is it the lack of N-glycosylation per se or an alteration in protein conformation caused by the changes in amino acid? Because almost all of the amino acids in this region are conserved, we undertook to prepare mutations in this region that only change the amino acids but do not block the N-glycosylation. Accordingly we prepared single mutations in the two N-glycosylation sites to change the middle Asn at position +1 to Gln (N536Q, N540Q) and the Thr at position +2 to Ser (T537S, T541S). After Endo H digestion, it was shown that all the mutants were glycosylated (Fig. 4E). Surprisingly, we found that none of the mutants showed any growth defect (Fig. 3B). Based on these findings, we conclude tentatively that N-glycosylation, rather than the amino acid sequence per se of these two N-glycosylation sites, is essential for the function of Stt3p. Mass Spectrometric Analysis Showed That Only One N-Glycosylation Site Was Utilized in Wild Type Stt3p—MS was utilized to determine whether one or both of the adjacent N-glycosylation sites were in fact glycosylated. Based on the MS result, only one glycan was found in this fragment, indicating that only one N-glycosylation site was utilized in the wild type Stt3p. Both a triply charged ion of m/z 1272.9 Da and a quadruply charged ion of m/z 954.6 Da were detected (data not shown). They correspond in molecular mass to the tryptic peptide TTLVDNNTWNNTHIAIVGK, with one high mannose-type glycan of the composition Man8GlcNAc2. This composition of an N-linked glycan is in agreement with the localization of Stt3p in the S. cerevisiae endoplasmic reticulum. The triply charged ion (1272.9) was subjected to MS/MS, and the gradual losses of 162 Da (the mass of a hexose) and 203 Da (N-acetylhexosamine) confirmed the glycan sequence (Fig. 7). No ions indicating the unglycosylated or doubly glycosylated peptide were detected. In addition, the tryptic peptide YLVNNSFYK containing the potential N-glycosylation site N60NS, which is not glycosylated, based on biochemical methods, was found only in the unglycosylated state (doubly charged ion, molecular mass of 574.68 Da) (data not shown), in agreement with the results obtained above. Because the tryptic peptide TTLVDNNTWNNTHIAIVGK contains two potential N-glycosylation sites, MS/MS/MS was used to sequence the peptide and determine the site of glycan attachment. The peptide was sequenced, and the data that were obtained indicate that the N-glycan is attached to the C-terminal-most N-glycosylation site in the peptide, that is, residue Asn539 (Fig. 8). Two doubly charged diagnostic peptides (y13 and y11) were found, along with other peptides resulting from fragmentation of this peptide. It is known that three of the essential subunits, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins in the yeast OT complex, but no information has been available regarding whether the N-glycosylation of these three subunits is important for OT activity. In addition, the extent of occupancy of the N-glycosylation sites in these proteins is not clear (1Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (171) Google Scholar). In this study, we first focused on these issues. We confirmed that all four potential sites on Ost1p and two potential sites on Wbp1p are occupied in the mature proteins, but none of these N-glycosylation sites is important for OT function. In Stt3p, there are three potential N-glycosylation sites, among which two (N535NT and N539NT) are highly conserved across the eukaryotic species and are separated by one amino acid. There were major growth defects and a loss of OT activity when the N-glycosylation of the conserved adjacent sites was blocked by single point mutations on either site or at both sites. However, mass spectrometric analysis showed that only the N539NT of the two adjacent sites was glycosylated in the wild type strain. It was not surprising to find that none of the N-glycosylation sites of Ost1p and Wbp1p was essential, because alignment showed that none of them is conserved among the eukaryotic species. However, two of the three N-glycosylation sites of Stt3p were highly conserved across eukaryotic species, implying that the N-glycosylation of these two sites and/or the amino acids in this region are important for Stt3p function. Our observations indicate that mutations in the N-glycosylation among the conserved sites result in cells exhibiting significant growth defects; they are either lethal or grow extremely slowly. However, when point mutations were prepared that altered the amino acid residues in the conserved sites but did not block the possibility of N-glycosylation, there was no effect on growth. Based on these results we conclude it is likely that the N-glycosylation per se is important for the function of Stt3p. Because the mutant proteins are still incorporated into the OT, the N-glycan chains are not required for interactions with the other proteins of the OT complex. Our current observations imply that the N-glycosylation of Stt3p is important for its function. However, what is the possible function for the glycans on Stt3p? Many studies have demonstrated the contribution of the glycan chains to protein folding, structure, and function (12Trombetta E.S. Glycobiology. 2003; 13: 77R-91RCrossref PubMed Scopus (168) Google Scholar). Analysis of the secondary structure around N-glycosylation sites suggests that the glycans are located at positions where changes in secondary structure occur, and some aromatic amino acids, which are distant from the N-glycosylation sites in the primary sequence, become close in the tertiary structure. These observations suggest that the N-glycans may participate directly in the folding of the protein as a consequence of glycan-protein interactions (13Petrescu A.J. Milac A.L. Petrescu S.M. Dwek R.A. Wormald M.R. Glycobiology. 2004; 14: 103-114Crossref PubMed Scopus (367) Google Scholar). In vitro experiments (14Nishimura I. Uchida M. Inohana Y. Setoh K. Daba K. Nishimura S. Yamaguchi H. J. Biochem. (Tokyo). 1998; 123: 516-520Crossref PubMed Scopus (27) Google Scholar, 15Jitsuhara Y. Toyoda T. Itai T. Yamaguchi H. J. Biochem. (Tokyo). 2002; 132: 803-811Crossref PubMed Scopus (47) Google Scholar, 16Mitra N. Sharon N. Surolia A. Biochemistry. 2003; 42: 12208-12216Crossref PubMed Scopus (36) Google Scholar) based on glycan-protein hydrophobic interactions also have provided evidence to support the concept that the glycans promote folding. In Stt3p, the predicted catalytic and/or recognition region containing two aromatic amino acids, W516W, is located approximately 20 amino acids N-terminal to the adjacent N-glycosylation sites, N535NTWN539NT. Based on two secondary structure prediction programs, nnPredict and PredictProtein, these two N-glycosylation sites are located on a loop that makes it flexible and possible for interaction with the catalytic site. Based on all of these results, we propose that the glycan at N539NT may be close to the catalytic site W516WDYG via the interaction between the glycan and the aromatic amino acids W516W (Fig. 1) and that this interaction may be important for the function of Stt3p. Finally, we asked wether the two N-glycosylation sites in Stt3p, which are separated by only one amino acid, are both glycosylated or whether only one site is glycosylated. Based on the Western blot, it was clear that when either N-glycosylation site was blocked, the other one was glycosylated, and, as expected, when both sites were blocked, there was no glycosylation. However, it is very difficult to determine whether both of the adjacent sites in Stt3p or only one site is glycosylated based on Western blot analysis. Therefore, we utilized mass spectrometry to answer this question. Interestingly, we found that only the N539NT site was glycosylated in the wild type Stt3p. However, when a similar double glycosylation site, NSTWNST, was engineered into preprolactin, the region was glycosylated on both Asn residues in 60% of the molecules during in vitro synthesis, in the presence of dog pancreas rough microsomes. 3I. Nilsson, personal communication. These observations suggest that a spatial constraint might exist to prevent the glycosylation of N535NT in Stt3p in vivo. Several decades ago, similar studies were performed in chicken ovalbumin, which has two N-glycosylation sites, Asn293 and Asn312, in which only the site Asn293 is glycosylated in vivo. However, both these sites are glycosylated in vitro (17Glabe C.G. Hanover J.A. Lennarz W.J. J. Biol. Chem. 1980; 255: 9236-9242Abstract Full Text PDF PubMed Google Scholar). In chicken ovalbumin, further experiments suggest that the absence of an oligosaccharide chain on Asn293 does not promote N-glycosylation of Asn312 (18Sheares B.T. J. Biol. Chem. 1988; 263: 12778-12782Abstract Full Text PDF PubMed Google Scholar). However, in Stt3p, N-glycosylation of N535NT is affected by the N-glycosylation of N539NT; and in wild type Stt3p, the N535NT is prevented from N-glycosylation. The unmodified amino acid Asn535 may be involved directly in some function of Stt3p, and this function may require N-glycosylation of Asn539. We propose that unglycosylated Asn535 serves a function that is lost when it is glycosylated because of the block of the glycosylation of N539NT. This model is consistent with the fact that all the mutants in this region are lethal or severely sick at 25 °C, and the Asn535 is glycosylated when Asn539 glycosylation site is blocked. It is also consistent with the fact that high conservation of the two adjacent sites is observed across a wide range of eukaryotic species. However, the mechanism may be different from archaeabacteria, which has been reported to have N-glycosylation activity (3Wacker M. Linton D. Hitchen P.G. Nita-Lazar M. Haslam S.M. North S.J. Panico M. Morris H.R. Dell A. Wren B.W. Aebi M. Science. 2002; 298: 1790-1793Crossref PubMed Scopus (628) Google Scholar). The homologue of Stt3p in Campylobacter jejuni does not have two adjacent N-glycosylation sites. It only has one N-glycosylation site, N534QS, after the predicted catalytic site, W457WDYG. In the current study, we found that 3 of the 10 N-glycosylation sites of the four OT subunits were not occupied (N175IS on Ost6p and N60NS and N535NT on Stt3p). Previous studies (17Glabe C.G. Hanover J.A. Lennarz W.J. J. Biol. Chem. 1980; 255: 9236-9242Abstract Full Text PDF PubMed Google Scholar, 19von Nilsson I.M. Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar, 20Popov M. Tam L.Y. Li J. Reithmeier R.A. J. Biol. Chem. 1997; 272: 18325-18332Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) indicated that N-glycosylation sites located less than 12–14 residues from a transmembrane segment of a protein are not modified in vivo, which also suggests that the active site of the OT is located 30–40 Å from the luminal face of the ER. In our case, the site of N175IS on Ost6p is located on the N terminus of the protein and is only 13 amino acids away from the N terminus of the first transmembrane domain. The N60NS on Stt3p is located on the first loop in the ER lumen, and it is 29 amino acids distant from the C terminus of the first transmembrane segment and 17 amino acids from the N terminus of the second transmembrane segment. This should be adequate space in primary sequence for N-glycosylation, and it is not clear why this site is not utilized. Statistical analysis indicated that not all potential N-glycosylation sites are occupied and that only 65% of the sites are glycosylated (13Petrescu A.J. Milac A.L. Petrescu S.M. Dwek R.A. Wormald M.R. Glycobiology. 2004; 14: 103-114Crossref PubMed Scopus (367) Google Scholar). It is particularly interesting that N535NT is not glycosylated in the wild type cells, whereas the N539NT site, which is separated from N535NT by only one amino acid, is glycosylated. We thought initially that the N-terminal-most N-glycosylation site N535NT would be favored for N-glycosylation. In fact, N535NT can be glycosylated when N539NT is blocked. But it is clear that this does not happen with wild type Stt3p. What causes the blockage and what function the conserved N535 serves remain unknown. We thank Dr. Reid Gilmore (University of Massachusetts Medical School) for anti-Ost1p and anti-Wbp1p antibodies, Dr. Randy Schekman (University of California, Berkeley) for anti-carboxypeptidase Y antibody, and Dr. Manasi Chavan for the microsomes with Ost6p tagged with 3HA. We also thank Drs. Gunnar von Heijne (University of Stockholm) and Robert Noiva (University of South Dakota) and members of Dr. Lennarz' laboratory for their comments on this work.
