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W2017807760 abstract "N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13–14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200–300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of α-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection. N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13–14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200–300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of α-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection. Protein glycosylation in eukaryotic cells is thought to play an essential role in many processes such as protein folding and transport, maintenance of protein and cell structure, and cell recognition and adhesion, as well as other functions. From the several types of protein glycosylation, N-glycosylation has received a great deal of attention not only because of its high frequency but also because several biochemical steps involved in this biosynthetic process are shared by yeast and humans, an indication that they have been conserved throughout evolution. These conserved steps occur in the membrane (i) or the lumen (ii and iii) of the ER 1The abbreviations used are: ER, endoplasmic reticulum; endo H, endoglicosidase H; HPLC, high pressure liquid chromatography; Exg1, exoglucanase. and belong to three groups: (i) assembly of the precursor oligosaccharide, GlnNAc2-Man9Glc3 on a lipid carrier (dolichol-PP), (ii) transfer of the oligosaccharide to the nascent or recently synthesized protein acceptor, and (iii) trimming of the three glucoses and one mannose (for recent reviews, see Refs. 1Orlean P. The Molecular Biology of the Yeast Saccharomyces. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 229-362Google Scholar and 2Burda P. Aebi M. Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar). However, once the glycoprotein leaves the ER, biochemical modification by trimming and/or addition of new sugars varies enormously between species and even between individual proteins of the same cell. This suggests that individual proteins carry the precise information for the final carbohydrate composition. In Saccharomyces cerevisiae, some of the protein-attached oligosaccharides leaving the ER (GlcNAc2-Man8) are poorly elongated with up to 13–14 mannoses (core-type), whereas many others are further elongated by the addition of an outer chain of up to 200 mannose residues in the Golgi apparatus (mannan-type), a process commonly known as hyperglycosylation. The outer chain consists of a backbone of α-1,6-mannoses with α-1,2 branches that are decorated with terminal α-1,3-mannose residues (1Orlean P. The Molecular Biology of the Yeast Saccharomyces. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 229-362Google Scholar, 3Ballou C.E. Methods Enzymol. 1990; 185: 440-470Crossref PubMed Scopus (275) Google Scholar). The biosynthesis of this complex is carried out by the ordered addition of mannoses in at least five biochemically defined steps (4Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (186) Google Scholar, 6Jungmann J. Rayner J.C. Munro S. J. Biol. Chem. 1999; 271: 6579-6585Abstract Full Text Full Text PDF Scopus (109) Google Scholar, 7Stolz J. Munro S. J. Biol. Chem. 2002; 277: 44801-44808Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) (Scheme 1). In S. cerevisiae, intracellular glycoproteins carry core-type oligosaccharides, whereas most extracellular glycoproteins carry outer chain-elongated structures. This suggests that hyperglycosylation may protect protein molecules from environmental constraints. It is well established that oligosaccharides are transferred from a lipid donor to specific asparagines in the tripeptide Asn-Xaa-(Ser/Thr), where Xaa is any amino acid except proline (8Marshall R.D. Biochem. Soc. Symp. 1974; 40: 17-26PubMed Google Scholar, 9Bause E. Lehle L. Eur. J. Biochem. 1979; 101: 531-540Crossref PubMed Scopus (59) Google Scholar, 10Bause E. Biochem. J. 1983; 209: 331-336Crossref PubMed Scopus (520) Google Scholar, 11Kaplan H.A. Welply J.K. Lennarz W.J. Biochim. Biophys. Acta. 1987; 906: 161-173Crossref PubMed Scopus (136) Google Scholar, 12Roitsch T. Lehle L. Eur. J. Biochem. 1989; 181: 525-529Crossref PubMed Scopus (78) Google Scholar). However, the structural principles that govern the frequency of glycosylation of the different sequons still are a matter of controversy. A survey of the N-linked sites has indicated that Thr functions better than Ser in this process (13Gavel Y. von Heijne G. Protein Eng. 1990; 3: 433-442Crossref PubMed Scopus (635) Google Scholar). Similarly, studies on glycosylation of the rabies virus glycoprotein have indicated that Ser containing sequons were poorly glycosylated in vitro relative to a similar series of sequons containing Thr in the third position (14Kasturi L. Eshleman J.R. Wunner W.H. Shakin-Eshleman S.H. J. Biol. Chem. 1995; 270: 14756-14761Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Also, work with yeast invertase showed that of the two overlapping sequons 4 and 5 of the protein (Asn92-Asn93-Thr94-Ser95), the first one (Thr) was almost completely glycosylated, but the second (Ser) was barely glycosylated, if at all (15Reddy V.A. Johnson R.S. Biemann K. Williams R.S. Ziegler F.D. Trimble R.B. Maley F. J. Biol. Chem. 1988; 263: 6978-6985Abstract Full Text PDF PubMed Google Scholar). Furthermore, a change in the tetrapeptide from NNTS to NNSS enabled both sequons to be glycosylated (16Reddy A. Gibbs B.S. Liu Y.L. Coward J.K. Changchien L.M. Maley F. Glycobiology. 1999; 9: 547-555Crossref PubMed Scopus (28) Google Scholar). However, the 100% glycosylation frequency in vivo of the Ser containing sequons present in the major yeast exoglucanase (Exg1) indicates that other parameters may have caused the bias in favor of Thr in the above mentioned studies or may have influenced our results (17Basco R.D. Hernández L.M. Muñoz M.D. Vázquez de Aldana C. Larriba G. Yeast. 1993; 9: 221-234Crossref PubMed Scopus (13) Google Scholar). Additional studies in vitro again using the rabies virus glycoprotein as a model have indicated that introduction of specific amino acids, such as Trp, Asp, Glu, or Leu, in the X position convert the sequon to a poor oligosaccharide acceptor (18Shakin-Eshleman S.H. Spitalnik S.L. Kasturi L. J. Biol. Chem. 1996; 271: 6363-6366Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 19Kasturi L. Chen H. Shakin-Eshleman S.H. Biochem. J. 1997; 323: 415-419Crossref PubMed Scopus (111) Google Scholar). Whereas studies on the identification of the structural features that influence the degree of occupation of a sequon are scarce and controversial, no attempts have been reported to specifically characterize hyperglycosylation. Gene fusions between either carboxypeptidase Y or proteinase A and invertase suggest that the proteases bear dominant signals that suppress hyperglycosylation of the invertase domain present in the fusion protein (20Johnson L.M. Bankaitis V.A. Emr S.D. Cell. 1987; 48: 875-885Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 21Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 2105-2116Crossref PubMed Scopus (190) Google Scholar). In an effort to identify determinants that regulate the extension of the N-oligosaccharide elongation, we have analyzed the effect of sequon composition on this process. Analysis of most extracellular glycoproteins is a difficult task. For this reason, we have used S. cerevisiae Exg1, an extracellular protein amenable to study that has been well characterized in our laboratory. Exg1 is classified in family 5 of glycosyl hydrolases (22Henrissat B. Davies G. Curr. Opin. Struct. Biol. 1997; 7: 637-644Crossref PubMed Scopus (1409) Google Scholar). In vivo Exg1 glycosylation yields several glycoforms. One of these, Exg1b, contains 12% carbohydrate distributed into two short oligosaccharides, each consisting of a regular inner core whose outer chain is reduced to two or three residues of mannose, indicating that the α-1,6-mannose added by Och1 is capped by a stop-signal α-1,2-mannose, which may be elongated with a terminal α-1,3-mannose (23Hernandez L.M. Olivero I. Alvarado E. Larriba G. Biochemistry. 1992; 31: 9823-9831Crossref PubMed Scopus (21) Google Scholar). These oligosaccharides are attached to both potential glycosylation sites (Asn165-Asn166-Ser167 and Asn325-Glu326-Ser327) present in the polypeptide (17Basco R.D. Hernández L.M. Muñoz M.D. Vázquez de Aldana C. Larriba G. Yeast. 1993; 9: 221-234Crossref PubMed Scopus (13) Google Scholar, 24Vázquez de Aldana C.R. Correa J. San Segundo P. Bueno A. Nebreda A.R. Méndez E. del Rey F. Gene. 1991; 97: 173-182Crossref PubMed Scopus (70) Google Scholar). Exg325 and Exg165 carry a single oligosaccharide attached to the second (Asn325) and the first (Asn165) glycosylation sites, respectively (17Basco R.D. Hernández L.M. Muñoz M.D. Vázquez de Aldana C. Larriba G. Yeast. 1993; 9: 221-234Crossref PubMed Scopus (13) Google Scholar). Exg1a contains 30–40% carbohydrate and forms smears in SDS-acrylamide gels as do other heavily glycosylated yeast glycoproteins (i.e. invertase, acid phosphatase), and its synthesis is prevented in mutant mnn9. Analysis of glycosylation mutants has indicated that only the second oligosaccharide of Exg1b can be elongated to generate Exg1a, an indication that it should be more accessible than the first one to the α-1,6-mannosyltransferase that elongates the outer chain (25Basco R.D. Hernández L.M. Muñoz M.D. Olivero I. Andaluz E. del Rey F. Larriba G. Biochem. J. 1994; 304: 917-922Crossref PubMed Scopus (9) Google Scholar). In this article we describe the effect of sequon composition, in particular the influence of the second amino acid of the tripeptide sequence (X) in the hyperglycosylation of Exg1, and we provide a structure-based hypothesis to explain our results. For this purpose, we have constructed mutated versions of the EXG1 gene in which the two sequons of the protein have been systematically mutated. Yeast Strains and Growth Conditions—Wild type S. cerevisiae TD28 (MATaura3–52 ino1–11 canr) and its Δexg1 derivative, CV55, have been described before (17Basco R.D. Hernández L.M. Muñoz M.D. Vázquez de Aldana C. Larriba G. Yeast. 1993; 9: 221-234Crossref PubMed Scopus (13) Google Scholar, 24Vázquez de Aldana C.R. Correa J. San Segundo P. Bueno A. Nebreda A.R. Méndez E. del Rey F. Gene. 1991; 97: 173-182Crossref PubMed Scopus (70) Google Scholar). S. cerevisiae YS57–5A (MATα, och1::LEU2 leu2 ura3 his1 his3) and S. cerevisiae BFY 109–1C (MATacan1–100 ade2–1 his3–11,-15 leu2–3,-112 trp1–1 ura3–1 kex2Δ::HIS3A) were kindly provided by Drs. Y. Jigami and R. Fuller, respectively. Yeast cells were maintained in YEPD medium (2% glucose, 1% yeast extract, 2% Bacto-peptone). For the production of external exoglucanase, cells were grown at 28 °C in liquid minimal medium supplemented with amino acids (26Hernandez L.M. Ramirez M. Olivero I. Larriba G. Arch. Microbiol. 1986; 146: 221-226Crossref PubMed Scopus (13) Google Scholar) until the middle exponential phase of growth. Plasmid Constructs—Centromeric plasmids carrying the EXG1 gene in pRS316 (27Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) (pRB1) and its derivatives in which the first (pRB2, EXG10E), the second (pRB3, EXG1N0), or both (pRB4, EXG100) sequons were eliminated by substituting the corresponding Asn by Gln have been described before (17Basco R.D. Hernández L.M. Muñoz M.D. Vázquez de Aldana C. Larriba G. Yeast. 1993; 9: 221-234Crossref PubMed Scopus (13) Google Scholar). All possible combinations between the X amino acids of the first and second sequons of Exg1 were performed by site-directed mutagenesis (QuikChange kit, Stratagene). First, using pRB1 as a template, a SalI/XbaI mutated fragment that included the first site as NES was obtained. It was then used to replace its counterpart in pRB1 and pRB4 to generate EXGEE and EXGE0, respectively. Similarly, a KpnI/ClaI mutated fragment including the second site as NNS was used to generate EXGNN and EXG0N from pRB1 and pRB4, respectively. Finally, EXGEN was obtained by introducing the SalI/XbaI mutated fragment into EXG0N. In a second set of experiments, we constructed all the possible variants of the second glycosylation site by introducing each one of other 19 amino acids (except proline) in the X position. For that purpose, the KpnI/NaeI fragment was amplified by PCR using suitable oligonucleotides that carry the appropriate mutation, and the resulting product was used to replace its counterpart in EXG00. All of the mutant constructs were confirmed by DNA sequencing. Purification and Characterization of Exg1—Culture supernatants obtained by centrifugation of cells were concentrated and dialyzed using Amicon PM10 membranes and/or Centricon filters. Purification of the different glycoforms of exoglucanase was carried out by ion exchange chromatography column (TSK gel DEAE-5PW, 7.5 mm × 7.5 cm, TosoHaas). Exoglucanase activity was determined using p-nitrophenol-β-d-glucopyranoside as a substrate (26Hernandez L.M. Ramirez M. Olivero I. Larriba G. Arch. Microbiol. 1986; 146: 221-226Crossref PubMed Scopus (13) Google Scholar). SDS-PAGE and Western blots were performed as reported (28Cueva R. Munoz M.D. Andaluz E. Basco R.D. Larriba G. Biochim. Biophys. Acta. 1996; 1289: 336-342Crossref PubMed Scopus (13) Google Scholar) using peroxidase to develop color. Standard deglycosylation reactions using endo H were carried out as described (25Basco R.D. Hernández L.M. Muñoz M.D. Olivero I. Andaluz E. del Rey F. Larriba G. Biochem. J. 1994; 304: 917-922Crossref PubMed Scopus (9) Google Scholar). (Endo H was a generous gift of Dr. F. Maley.) Exg1 Structure Analysis—Crystal structure of the S. cerevisiae exo-1,3-β-exoglucanase has been determined recently (29Taylor S.C. Ferguson A.D. Bergeron J.J. Thomas D.Y. Nat. Struct. Mol. Biol. 2004; 11: 128-134Crossref PubMed Scopus (112) Google Scholar). Atomic coordinates were retrieved from the Protein Data Bank (Protein Data Bank accession code 1H4P). Protein structure was analyzed with a Swiss-Model and Swiss-Pdb Viewer (30Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9589) Google Scholar, 31Guex N. Diemand A. Peitsch M.C. Trends Biochem. Sci. 1999; 24: 364-367Abstract Full Text Full Text PDF PubMed Google Scholar). Analysis of the Exg1 Glycoforms Secreted by S. cerevisiae Expressing Mutated Versions of EXG1 with Altered Sequon Composition—Wild type EXG1 or each one of the EXG1 constructs carrying all possible combinations of the first and second sequons was cloned in the centromeric plasmid PRS316 (27Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). These clones were then used to transform strain CV55 (Δexg1), and the transformants were grown at 28 °C for 15 h. The supernatant fluids, dialyzed and concentrated, were fractionated by ion exchange chromatography (HPLC). A summary of the nature and amount of the several glycoforms secreted by ectopic wild type EXG1 and glycosylation mutants is shown in Table I. Wild type EXG1 showed the typical profile, a minor and heterogeneous peak (Exg1a, 10%) preceded a major and sharp one (Exg1b, 90%) (Fig. 1A, panel a). Glycosylation mutants in which one sequon had been eliminated also yielded the expected results. Thus, mutant N0 only generated Exg165, whereas mutant 0E yielded an Exg1a-like glycoform and Exg325. Western blot analysis of Exg1b, Exg165, and Exg325 confirmed the nature of these forms indicating that the length of the short oligosaccharides attached to the modified sites was not significantly altered (Fig. 2). When the second sequon was eliminated (N325Q) and the first one was constructed NES (notice that this corresponds to the second sequon in native Exg1) (form E0), an Exg165-like enzyme was secreted indicating that the change introduced in the amino acid X of the first sequon (Asn to Glu) does not modify the elongation properties of the attached oligosaccharide. However, when the first sequon was eliminated (N165Q) and the second one was NNS (notice that Asn corresponds to the first sequon in native Exg1) (form 0N), only about 70% of the activity eluted as an Exg325-like form, whereas the residual 30% eluted as two peaks in the Exg1a region (Table I and Fig. 1A, panel c). This result suggested that the presence of Asn instead of Glu in the X residue of the second sequon increases the probabilities for elongation of the attached oligosaccharide. We should emphasize that because purified Exg1a and Exg1b have the same specific activity (32Ramírez M. Hernández L.M. Larriba G. Arch. Microbiol. 1989; 151: 391-398Crossref PubMed Scopus (28) Google Scholar), the enzymatic activity exhibited by each glycoform can be taken as a good estimation of the amount of the associated Exg1p (see also below).Table IA summary of the nature and amount of the Exg1p glycoforms secreted by the indicated glycosylation mutantsExg1 variantFirst sequonSecond sequonAssociated glycoformsExgNENNSNESExg1a (10%) + Exg1bExgEENESNESExg1a (10%) + Exg1bExgNNNNSNNSExg1a (30%) + Exg1bExgENNESNNSExg1a (30%) + Exg1bExgE0NESQESExg165-likeExg0NQNSNNSExg1a-like (30%) + Exg325ExgN0NNSQESExg165Exg0EQNSNESExgla-like (10%) + Exg325Exg00QNSQESNone Open table in a new tab Fig. 2Time course deglycosylation of the hyperglycosylated variant produced by construct NN.A, analysis in HPLC. Panel a, isolated hyperglycosylated fraction. Panels b–g, hyperglycosylated fractions following treatment with endo H for 1.5, 3, 6, 12, 22, and 40 h, respectively. B, analysis by Western blots. C, quantification of substrates and reaction products from the HPLC eluate.View Large Image Figure ViewerDownload (PPT) Mutant constructs with two glycosylation sites confirmed these observations and added new data (Table I). Thus, the EE construct (both sequons are identical to the second sequon of the wild type exoglucanase) generated an exoglucanase complement indistinguishable from the wild type counterpart by both HPLC (Fig. 1A) and SDS-PAGE (Fig. 2), indicating that the presence of Glu instead Asn at the X position of the first sequon does not alter the glycosylation pattern of wild type Exg1. On the other hand, the EN construct (both sequons exchange their positions) yielded two peaks in the Exg1a region (30%) and one eluting as Exg1b (70%) (Table I and Fig. 1A, panel b), the latter being further characterized by Western blotting as a form carrying two short oligosaccharides (Fig. 2). Finally, mutant NN (both sequons are identical to the first sequon of wild type exoglucanase) behaved as its EN counterpart (Table I and Fig. 2). Therefore, in all three constructs carrying Asn at the X position of the second sequon (0N, EN, and NN), there is a significant increase in the amount of exoglucanase activity in the Exg1a region (30%) as compared with wild type (10%) (Fig. 1A). The absence of subglycosylated and/or non-glycosylated forms in transformants expressing wild type EXG1 or mutant constructs with two glycosylation sites as well as the absence of non-glycosylated Exg in transformants carrying constructs N0, 0E, E0, and 0N indicates that the transfer of oligosaccharides is very efficient and that there is enough lipid-linked oligosaccharide available to occupy all the sites offered by the nascent exoglucanase during its translocation into the lumen of the ER. It should be noticed that regardless of their position in the molecule the QES sequon is always efficiently core-glycosylated. This contrasts with the results obtained with a variant of the rabies virus glycoprotein, which have indicated that the presence of Glu at the X position is associated with inefficient core glycosylation (18Shakin-Eshleman S.H. Spitalnik S.L. Kasturi L. J. Biol. Chem. 1996; 271: 6363-6366Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and suggests that other protein signals must also control this process. Characterization of the Hyperglycosylated Forms from ON, EN, and NN Constructs—Although mutant exoglucanases eluting in the ExgIa region seem to correspond to hyperglycosylated forms, the fact that an immature form of exoglucanase (form A) also elutes in this region prompted us to distinguish these possibilities. Form A is an endoplasmic reticulum form of Exg1 carrying a 21-amino acid propeptide; it is converted to mature form in the Golgi apparatus by elimination of the propeptide by the Kex2 protease (33Basco R.D. Giménez-Gallego G. Larriba G. FEBS Lett. 1990; 268: 99-102Crossref PubMed Scopus (19) Google Scholar, 34Basco R.D. Cueva R. Andaluz E. Larriba G. Biochim. Biophys. Acta. 1996; 1310: 110-118Crossref PubMed Scopus (9) Google Scholar). Although Exg1a and form A co-eluted in HPLC, their deglycosylated products have quite different retention times. As shown in Fig. 1A (top of graph), following treatment with endo H, authentic form A is converted into a deglycosylated product eluting in HPLC as native ExgIb (fraction 19), whereas authentic form B yields a much more acidic compound (fraction 30). Similarly, after treatment with endo H, Exg1a from construct EN was quantitatively converted into a form undistinguishable from deglycosylated ExgIb. The absence of deglycosylated form A indicates that all of the exoglucanase eluting in the Exg1a region is indeed mature and hyperglycosylated. The same was true for the Exg1a-like counterparts generated by constructs EE and NN. As expected, deglycosylation of the Exg1a-like form generated by construct 0N yielded a product that co-eluted with the deglycosylated product of the Exg325-like exoglucanase generated by the same construct. These biochemical data were also supported by genetic evidence. Oligosaccharides from glycoproteins secreted by mutant Δoch1 are unable to elongate the inner core, but the protein portion of susceptible substrates (α factor or Exg1) are normally processed; accordingly, this mutant only secretes an Exg1b-like form with no traces of ExgIa. YS57–5A cells (Δoch1) transformed with the EN construct exclusively secreted Exg1b. Because the Δoch1 mutation does not prevent secretion of form A in a Δkex2 mutant (data not shown), we conclude that the Exg1a-like enzymes under study do not correspond to form A, but instead they behave as hyperglycosylated forms of mature exoglucanase. In agreement with its hyperglycosylated nature, all of the Exg1a-like forms yielded by constructs NN, EN, and 0N smeared when analyzed in SDS-PAGE where they exhibited a similar molecular size (Fig. 1C, panel a). Obviously, the absence of the first short oligosaccharide (Mr 3000) in the 0N constructs is not enough to introduce detectable differences in the upper size limit. This observation indicates that the absence of the first oligosaccharide does not influence the degree of elongation of the second. It should also be noted that hyperglycosylated forms derived from constructs EN, NN, and 0N elute clearly into two peaks, a property that we have extended now to wild type Exg1a where it was not as evident because of the low levels of this glycoform. Interestingly, as shown for the construct 0N (Fig. 1C, panel b), the Exg1 molecules included in peak 1 have an average size larger than their counterparts from peak 2, indicating that in the former the oligosaccharide attached to the second site carries a more elongated outer chain. Although the precise origin and nature of these differences is under study, they likely are derived from different rounds of action of mannan polymerase I, mannan polymerase II, or both. To further investigate the nature of the Exg1a-like enzyme secreted by NN and EN transformants, we purified the whole hyperglycosylated fraction by preparative HPLC and subjected it to treatment with endo H. The results were highly reproducible and almost identical for both transformants; accordingly, we will present only those results from construct NN. Time course deglycosylation was followed by both ion exchange chromatography (Fig. 2A) and Western blots (Fig. 2B). The deglycosylation kinetics in terms of the enzymatic activity associated with the several glycoforms (Exg1a, peak 1 and peak 2; Exg1b, subglycosylated and unglycosylated Exg1) is shown in Fig. 2C. The following conclusions can be derived. (i) As expected, the hyperglycosylated exoglucanase was quantitatively transformed into deglycosylated Exg1, which carries one GlcNAc attached to each sequon. The absence of more acidic products indicates that all the sequons of the hyperglycosylated molecules carried oligosaccharides. The final product of the reaction, deglycosylated Exg1, was also detected in Western blots as a 47-kDa band. (ii) A deglycosylation intermediate (fraction 27) is produced during the first stages of the deglycosylation reaction. Then, its levels decreased, and it was no longer visible after the levels of the substrate dropped to one-half. This intermediate eluted in the same position as the glycoform with a short oligosaccharide in the first position and a single GlcNAc in the second one and was also identified in the same samples by Western blots as a 50-kDa band; these features make it indistinguishable from the second intermediate detected during the deglycosylation of Exg1b (17Basco R.D. Hernández L.M. Muñoz M.D. Vázquez de Aldana C. Larriba G. Yeast. 1993; 9: 221-234Crossref PubMed Scopus (13) Google Scholar). The most likely explanation is that the intermediate arises from the hyperglycosylated forms by elimination of the elongated residue. The levels of this intermediate are always very low because the endoglycosidase has more affinity for the oligosaccharide attached to the first position in such a way that not only it is produced at low levels, but it is also immediately transformed into deglycosylated enzyme (25Basco R.D. Hernández L.M. Muñoz M.D. Olivero I. Andaluz E. del Rey F. Larriba G. Biochem. J. 1994; 304: 917-922Crossref PubMed Scopus (9) Google Scholar). (iii) Both peaks of the hyperglycosylated region behaved similarly, although the deglycosylation kinetics of peak 1 was slightly faster. The preference of endo H for the first (short) oligosaccharide suggests the generation of an abundant intermediate carrying the elongated oligosaccharide attached to the second sequon. This intermediate could not be detected in the chromatograms indicating that its elution time is similar that of its precursor. Deglycosylation of the Exg1a-like enzyme from the 0N transformant yielded similar results, except that there were no traces of the small intermediate (see Fig. 2). These results unambiguously demonstrate that the mutant glycoforms eluting in the Exg1a area in the HPLC column truly correspond to hyperglycosylated exoglucanase. Therefore, we conclude that substitution of Glu by Asn at the X position of the second sequon significantly increases the hyperglycosylation efficiency. Analysis of the Hyperglycosylation of Exg1 Variants with Amino Acid Substitution at the X Position of Sequon 2—To further analyze how hyperglycosylation is affected by the nature of the second amino acid of the sequon, we systematically changed the amino acid at position X of the second glycosylation site. To facilitate the analysis, these constructs were placed in a context in which the first glycosylation site had been eliminated (N165Q). As shown in Fig. 3A, analysis of the secreted Exg1 glycoforms derived from the new constructs indicated the presence of a substantial amount (up to 57%) of hyperglycosylated Exg1 when the" @default.
W2017807760 created "2016-06-24" @default.
W2017807760 creator A5006267287 @default.
W2017807760 creator A5033190295 @default.
W2017807760 creator A5036027102 @default.
W2017807760 creator A5058020739 @default.
W2017807760 creator A5085307609 @default.
W2017807760 date "2004-10-01" @default.
W2017807760 modified "2023-09-30" @default.
W2017807760 title "A Search for Hyperglycosylation Signals in Yeast Glycoproteins" @default.
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