FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2037439168> ?p ?o ?g. }

• W2037439168 endingPage "195" @default.
• W2037439168 startingPage "183" @default.
• W2037439168 abstract "Nascent and newly synthesized glycoproteins enter the calnexin (Cnx)/calreticulin (Crt) cycle when two out of three glucoses in the core N-linked glycans have been trimmed sequentially by endoplasmic reticulum (ER) glucosidases I (GI) and II (GII). By analyzing arrested glycopeptides in microsomes, we found that GI removed the outermost glucose immediately after glycan addition. However, although GII associated with singly glycosylated nascent chains, trimming of the second glucose only occurred efficiently when a second glycan was present in the chain. Consistent with a requirement for multiple glycans to activate GII, pancreatic RNase in live cells needed more than one glycan to enter the Cnx/Crt cycle. Thus, whereas GI trimming occurs as an automatic extension of glycosylation, trimming by GII is a regulated process. By adjusting the number and location of glycans, glycoproteins can instruct the cell to engage them in an individually determined folding and quality control pathway. Nascent and newly synthesized glycoproteins enter the calnexin (Cnx)/calreticulin (Crt) cycle when two out of three glucoses in the core N-linked glycans have been trimmed sequentially by endoplasmic reticulum (ER) glucosidases I (GI) and II (GII). By analyzing arrested glycopeptides in microsomes, we found that GI removed the outermost glucose immediately after glycan addition. However, although GII associated with singly glycosylated nascent chains, trimming of the second glucose only occurred efficiently when a second glycan was present in the chain. Consistent with a requirement for multiple glycans to activate GII, pancreatic RNase in live cells needed more than one glycan to enter the Cnx/Crt cycle. Thus, whereas GI trimming occurs as an automatic extension of glycosylation, trimming by GII is a regulated process. By adjusting the number and location of glycans, glycoproteins can instruct the cell to engage them in an individually determined folding and quality control pathway. The majority of proteins synthesized in the ER are glycoproteins. In fact, almost half of the genes in the human genome are thought to encode for proteins linked to sugars, and most of these contain N-linked oligosaccharides (Apweiler et al., 1999Apweiler R. Hermjakob H. Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.Biochim. Biophys. Acta. 1999; 1473: 4-8Crossref PubMed Scopus (1392) Google Scholar). N-glycans confer increased stability, solubility, and protection to proteins. They also engage in recognition events in a large variety of cellular processes. Although N-linked glycans in mature glycoproteins display extensive heterogeneity due to processing in the Golgi complex, the structure of the core oligosaccharide added to nascent polypeptide chains in the ER by oligosaccharyl transferase (OST) is relatively simple. It is a branched structure composed of two N-acetylglucosamines, nine mannoses, and three glucoses (Glc3Man9GlcNAc2) shared by virtually all eukaryotes (Figure 1A ). Over the past 10 years, research has focused on the role of the terminal glucose and mannose residues that undergo trimming by glycosidases in the ER. It has become clear that partially trimmed glycans serve as signals during glycoprotein folding and quality control (Dejgaard et al., 2004Dejgaard S. Nicolay J. Taheri M. Thomas D.Y. Bergeron J.J. The ER glycoprotein quality control system.Curr. Issues Mol. Biol. 2004; 6: 29-42PubMed Google Scholar, Helenius and Aebi, 2004Helenius A. Aebi M. Roles of N-linked glycans in the endoplasmic reticulum.Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1518) Google Scholar). Importantly, trimming of glucoses allows nascent and newly synthesized glycoproteins to enter into and escape from the Cnx/Crt cycle, a chaperone system that supports glycoprotein maturation and quality control in the ER. Cnx and Crt are lectins that bind to monoglucosylated intermediates of the core glycan. The key trimming enzymes in the ER are GI, GII, and ER mannosidase I. GI removes the outermost of the three glucoses, and GII is responsible for removing the middle glucose (cleavage 1). The monoglucosylated glycans thus generated allow newly synthesized glycoproteins to bind to Cnx and Crt. The release of the glycoprotein from these chaperones involves a second GII cleavage (cleavage 2), which removes the remaining glucose. If a glycoprotein is properly folded after release from the cycle, it is free to leave the ER. If not, it is reglucosylated by UDP-Glc: glycoprotein glucosyl transferase, which acts as a folding sensor driving the improperly folded protein back into association with Cnx and Crt (Sousa et al., 1992Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (262) Google Scholar). In this way, glycoproteins can go through multiple rounds of interaction with the chaperones. If they still fail to fold properly, the slow-acting ER mannosidase I will cleave the terminal mannose in the B branch, thereby targeting the glycoprotein for degradation. To characterize the glucose-trimming events and their role in determining substrate flux into the Cnx/Crt cycle, we addressed the activities of GI and GII in microsomes and live cells. GI is a type II membrane glycoprotein with a lumenal catalytic domain (Shailubhai et al., 1991Shailubhai K. Pukazhenthi B.S. Saxena E.S. Varma G.M. Vijay I.K. Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain.J. Biol. Chem. 1991; 266: 16587-16593Abstract Full Text PDF PubMed Google Scholar), and GII is a soluble heterodimer. In addition to the catalytic α chain (GIIα), GII contains a noncovalently bound β chain (GIIβ) (Trombetta et al., 1996Trombetta E.S. Simons J.F. Helenius A. Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit.J. Biol. Chem. 1996; 271: 27509-27516Crossref PubMed Scopus (200) Google Scholar). Our results show that glucose trimming by GI is a rapid and efficient process that occurs immediately after addition of a glycan to the polypeptide chain. They also reveal that trimming of the middle glucose by GII is a carefully regulated process. It provides cells with a sophisticated mechanism to modulate the entry of nascent and newly synthesized glycoproteins to the Cnx/Crt cycle. The number of N-linked glycans emerges as an important determinant for chaperone interactions in the ER and ultimately glycoprotein fate. To determine how far into the lumen of the ER a peptide bound glycan must move before it is accessed by the trimming enzymes, we produced glycopeptides of different length by in vitro translation in ER-derived rough microsomes. Microsomes not only support cotranslational translocation of nascent polypeptides but also proper glycosylation and glycan trimming (Marquardt et al., 1993Marquardt T. Hebert D.N. Helenius A. Post-translational folding of influenza hemagglutinin in isolated endoplasmic reticulum-derived microsomes.J. Biol. Chem. 1993; 268: 19618-19625Abstract Full Text PDF PubMed Google Scholar). To generate translation-arrested chains that mimic translation intermediates, we used truncated mRNAs. When a ribosome reaches the 3′ end of an mRNA devoid of a termination codon, it stalls, and the translated polypeptide remains trapped in the ribosome as a peptidyl-tRNA (Gilmore et al., 1991Gilmore R. Collins P. Johnson J. Kellaris K. Rapiejko P. Transcription of full-length and truncated mRNA transcripts to study protein translocation across the endoplasmic reticulum.Methods Cell Biol. 1991; 34: 223-239Crossref PubMed Scopus (72) Google Scholar). By varying the length of the mRNA, we could position the glycosylation sequons and N-linked glycans at defined locations in relation to the translocon complex and OST. To detect small changes in molecular weight resulting from glucose trimming, the arrested chains had to be as short as possible, and hence, the glycosylation site of interest close to the N terminus. To avoid inefficient glycosylation of sequons close to the signal peptide, we made use of the Semliki Forest virus (SFV) capsid protease domain. It has the capacity to fold and cleave itself off cotranslationally as soon as it emerges from the translocon complex (Kowarik et al., 2002Kowarik M. Kung S. Martoglio B. Helenius A. Protein folding during cotranslational translocation in the endoplasmic reticulum.Mol. Cell. 2002; 10: 769-778Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In this way, the growing nascent chains downstream of the cleavage site are subjected to equivalent conditions regardless of their size. Our model protein consisted of SFV p62 glycoprotein, preceded by the capsid protease domain and the signal sequence of vesicular stomatitis virus glycoprotein (Figure 1B). p62 contains two glycosylation sites at Asn12 and 59. We used either this construct (NN) or a construct in which glycosylation site 59 was eliminated (N). Both served as templates to generate a panel of truncated mRNAs coding for arrested chains ranging from 72 to 168 amino acids in length. The nomenclature adopted for the peptides gives the number of amino acids that separates each of the glycosylation sites from the ribosomal P site. For example, an arrested chain 112 amino acids long was named p100/53 in NN and p100 in N (see Figure 1B and Figure S1 available in the Supplemental Data with this article online). To span the distance between the P site and the OST active site, 65–75 amino acids are required depending on the tendency of the nascent chain to adopt an α helix in the ribosome-translocon channel (Mingarro et al., 2000Mingarro I. Nilsson I. Whitley P. von Heijne G. Different conformations of nascent polypeptides during translocation across the ER membrane.BMC Cell Biol. 2000; 1: 3Crossref PubMed Scopus (70) Google Scholar). Because this is a wide window, we first determined the number of amino acids required for glycosylation of the p62 peptides. A panel of truncated mRNAs was translated in the presence of 35S-Met. The microsomes were isolated, and the translocated proteins separated by SDS-PAGE using a gel system with high resolution in the low molecular weight range. After autoradiography, several labeled bands were observed (Figure 2A ). In addition to a band of constant size corresponding to the 19 kDa capsid protease domain (marked by an asterisk in this and subsequent gels), there were two series of bands of decreasing mobility with increasing peptide size. Whereas bands belonging to the faster migrating ladder were present in all lanes, bands in the slower set were detected only in p68/21 and longer peptides (arrowhead, lanes 3–8). Upon treatment with PNGase F, the slower migrating bands disappeared, and the intensity of the faster migrating bands increased (Figure 2B), demonstrating that they corresponded to the glycosylated and nonglycosylated peptides, respectively. When the arrested nascent chains were released from the P site by treating the microsomes with puromycin, a fraction of the peptides in which the Asn12-P distance is shorter than 68 residues was also glycosylated (Figure 2C). Moreover, a third, even slower migrating ladder appeared corresponding to doubly glycosylated chains (upper arrow). The same pattern was seen when the mRNAs had a stop codon for proper chain termination (Figure S2). By using longer arrested chains, cotranslational glycosylation of the second site was first detected for peptide p112/65, i.e. when Asn59 was 65 residues from the P site (Figure 2E). To analyze the fate of the Asn12 glycan alone, some of the experiments below made use of mRNAs derived from the N construct (Figure S3). Quantification determined that half-maximal glycosylation of both Asn12 and Asn59 was reached when they were located 72 residues from the P site (Figure 2D). In summary, short peptides with 0, 1, or 2 N-linked glycans could be generated in microsomes. When translated from truncated mRNAs, they were arrested in the translocon complex, allowing glycosylation only when the chains were long enough to present their glycosylation sites to OST. To make the removal of the outermost glucose residue by GI detectable in SDS-PAGE, we took advantage of the fact that GI trimming renders the two remaining glucoses sensitive to purified GII (Figure 3A ). Removal of the two glucoses, in turn, makes eight of the nine mannoses sensitive to jack bean α-mannosidase (α-man), an exomannosidase that cleaves mannoses from nonreducing termini. In glycans that have three glucose residues, only five mannoses are removed by α-man. The resulting mobility difference in SDS-PAGE was used to distinguish untrimmed glycopeptides from those trimmed by GI. To determine how far from the P site a peptide bound glycan had to be to have access to GI, translation was performed for different times in the presence and the absence of castanospermine (Cst), a GI and GII inhibitor. The microsomes were isolated and solubilized with cholate to maintain the interaction between the ribosomes and the arrested chains while removing the components of the translocation machinery (Matlack and Walter, 1995Matlack K.E. Walter P. The 70 carboxyl-terminal amino acids of nascent secretory proteins are protected from proteolysis by the ribosome and the protein translocation apparatus of the endoplasmic reticulum membrane.J. Biol. Chem. 1995; 270: 6170-6180Crossref PubMed Scopus (52) Google Scholar). The ribosome-arrested chain complexes were collected by centrifugation, digested with purified GII, treated with α-man, and analyzed by SDS-PAGE and autoradiography. In this way, only 35S-Met-labeled nascent chains still attached to the ribosomes could be visualized. We performed the analysis by using peptide p76, which was barely long enough to reach maximal glycosylation of Asn12 (see Figure 2D). Samples containing Cst during translation were used as markers for triglucosylated glycopeptides. Already after 5 min, 61% of the glycosylated chains were converted to the faster migrating GI-trimmed form, reaching maximal trimming after 4 min (Figures 3B and 3C). Considering the translation rate in the in vitro system (Ujvari et al., 2001Ujvari A. Aron R. Eisenhaure T. Cheng E. Parag H.A. Smicun Y. Halaban R. Hebert D.N. Translation rate of human tyrosinase determines its N-linked glycosylation level.J. Biol. Chem. 2001; 276: 5924-5931Crossref PubMed Scopus (67) Google Scholar), 5 min are just enough to finish the synthesis of the chains that were initiated first. This indicates that the N-glycan in p76 is immediately accessible to GI. We therefore analyzed a shorter peptide, p72, which only achieved half-maximal glycosylation (Figure 2D). As shown in Figures 3C and 3D, p72 was also processed by GI, although more slowly than p76, with half-maximal trimming reached after 9 min. The results indicated that GI has access to the core glycan as soon as it has been added to the polypeptide chain. This suggests that GI must be closely associated with the translocon complex and/or the OST. To determine how far from the P site a glycan had to be for GII cleavage to occur, we analyzed a series of arrested singly glycosylated peptides of increasing length. To be able to differentiate between peptides that carry a glycan with 0, 1, or 2 glucoses, we generated a set of marker glycopeptides by sequential trimming in vitro (see Experimental Procedures). The markers were run along with our samples in SDS-PAGE. We found to our surprise that GII did not trim any of the peptides (Figure 4A , and Figure S4 for a peak composition analysis), not even the longest, p104, in which the glycan was located ∼32 residues beyond the OST active site. We tested GII enzymatic activity directly and found that the microsomes contained 0.05 mU of GII per membrane equivalent. This corresponded to a 10-fold higher concentration of purified GII than that of our standard in vitro assays (i.e., Figure 3). Because Cnx only recognizes monoglucosylated glycoproteins, i.e., products of the first GII cleavage step, we determined whether any of the chains interacted with Cnx. Consistent with the lack of GII trimming, immunoprecipitation (IP) with α-Cnx antibodies failed to coprecipitate any peptide up to p156 (Figure 4B). The same results were obtained with arrested chains containing a single glycosylation site at Asn59 (Figures S5 and S6). Binding to Crt was also not observed when using α-Crt antibodies (see Figure 5B). These results showed that, although abundantly present in the microsomes, GII was unable to process a glycan in arrested glycopeptides located as far as ∼84 amino acids from the OST active site (i.e., Ans12 in p156). This was rather unexpected, as Cnx and Crt have been shown to interact with growing nascent glycopeptide chains in cells and microsomes (e.g., Daniels et al., 2003Daniels R. Kurowski B. Johnson A.E. Hebert D.N. N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin.Mol. Cell. 2003; 11: 79-90Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Hebert et al., 1997Hebert D.N. Zhang J.X. Chen W. Foellmer B. Helenius A. The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin.J. Cell Biol. 1997; 139: 613-623Crossref PubMed Scopus (210) Google Scholar, and Molinari and Helenius, 2000Molinari M. Helenius A. Chaperone selection during glycoprotein translocation into the endoplasmic reticulum.Science. 2000; 288: 331-333Crossref PubMed Scopus (278) Google Scholar). Could the lack of trimming mean that arrested glycopeptide chains were somehow protected from GII while associated with the translocon? To answer this question, we tested whether release of the glycopeptides into the microsomal lumen by puromycin would lead to trimming by GII. To monitor the effectiveness of the puromycin-induced release, we used in this experiment p76/29, a peptide in which Asn12 was glycosylated cotranslationally, whereas Asn59 was only accessible to OST after chain release. As expected, without puromycin the peptides had either one or no glycan (Figure 4C, lanes 1–2). When Cst was present during translation (lane 1), the chains with a single glycan had a slightly slower migration than without Cst (lane 2), confirming that GI was inhibited. If puromycin was added after translation, a third band appeared corresponding to p76/29 glycosylated in both sites (lanes 3–4, upper arrowheads), indicating that chain release had occurred. We noticed, however, that the mobility of the doubly glycosylated p76/29 was strikingly different between samples treated with Cst or not. Although the untrimmed version of the chain in the presence of Cst migrated as a sharp band (lane 3), the corresponding band in the absence of Cst was broad and had a clearly faster mobility (lane 4). This suggested that the doubly glycosylated chains had not only been trimmed by GI but also by GII. That p76/29 with two glycans was trimmed by GII was confirmed by Co-IP with α-Cnx antibodies (Figure 4C). Doubly glycosylated p76/29 coprecipitated specifically with the lectin (lane 8), whereas chains with one or no glycan coprecipitated with Cnx nonspecifically and at background levels (compare lanes with and without Cst). Because Cnx only binds to chains with monoglucosylated glycans, this implied that at least one of the glycans in the doubly glycosylated p76/29 must have been subjected to GII trimming. A weaker band with the mobility of doubly glycosylated, GII-trimmed p76/29, was also seen in α-Cnx precipitates from samples without Cst or puromycin (lane 6). Most likely, these GII-trimmed peptides represented a fraction that was spontaneously released from the ribosomes, a process known to occur in microsomes (Gilmore et al., 1991Gilmore R. Collins P. Johnson J. Kellaris K. Rapiejko P. Transcription of full-length and truncated mRNA transcripts to study protein translocation across the endoplasmic reticulum.Methods Cell Biol. 1991; 34: 223-239Crossref PubMed Scopus (72) Google Scholar). Evidently, the doubly glycosylated chains released into the ER lumen were rapidly trimmed by GII and entered the Cnx cycle. To test whether trimming was due to the number of glycans or to the release of glycopeptides into the lumen, we analyzed the peptide p144/97. This peptide was long enough to acquire two glycans while ribosome bound. We compared it with p144, a peptide of the same length with a single glycosylation site. Both peptides were analyzed in arrested and puromycin-released forms. As shown in Figure 5A , the singly glycosylated p144 was not efficiently trimmed by GII and failed to coprecipitate with Cnx regardless of puromycin-induced release. In contrast, the doubly glycosylated p144/97 showed a large mobility shift and coprecipitated significantly with Cnx. This occurred whether p144/97 was translocon associated or released by puromycin (quantifications in Figure 5B). We concluded that efficient trimming by GII and subsequent binding to Cnx were determined by the number of glycans in a chain. More than one was needed to observe GII cleavage 1. To rule out the possibility that there was a significant difference in affinity of Cnx for chains with a single versus multiple glycans, we generated peptides with a single monoglucosylated glycan. To force GII trimming of glycopeptides with one glycan, we took advantage of an observation by Popov and Reithmeier, 1999Popov M. Reithmeier R.A. Calnexin interaction with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger 1 (band 3).J. Biol. Chem. 1999; 274: 17635-17642Crossref PubMed Scopus (24) Google Scholar. They found that a singly glycosylated protein can enter a Cnx complex if released into the ER lumen with puromycin and incubated for an extended time. We used p80 because it was short enough to allow the trimming status to be easily determined by SDS-PAGE. After 30 min incubation, a fraction of the glycopeptide had undergone extensive trimming (Figure 5C, lanes 3 and 4, peak composition in Figure S7), and ∼5% coprecipitated with Cnx (lanes 7 and 8, quantifications in Figure S8). This indicated that p80 had undergone GII cleavage 1, demonstrating that Cnx can bind singly glycosylated peptides if such chains occur in the microsomes. When puromycin was omitted from the samples, GII trimming and Cnx binding were largely inhibited (Figure 5C, right). The small amount of monoglucosylated peptides observed after 60 min (lane 12) was likely due to time-dependent spontaneous release of chains (Gilmore et al., 1991Gilmore R. Collins P. Johnson J. Kellaris K. Rapiejko P. Transcription of full-length and truncated mRNA transcripts to study protein translocation across the endoplasmic reticulum.Methods Cell Biol. 1991; 34: 223-239Crossref PubMed Scopus (72) Google Scholar). The result suggested that singly glycosylated chains could be trimmed by GII when they were released into the ER lumen, but not so if they remained arrested in the translocon. To determine how far the second glycan had to move into the lumen of the microsome before GII trimming occurred, we analyzed Cnx binding to a series of peptides of increasing length (Figure 5D). Of p116/69, one of the shortest peptides to have acquired two glycans (see Figure 2D), ∼8% were coprecipitated with Cnx. For the longer chains, coprecipitation increased slightly, reaching mean values of ∼12%. This indicated that as soon as the second glycan was added to the chain by OST and trimmed by GI, the glycopeptide became a substrate for GII cleavage 1. The immediate access of GII to the second glycan suggested that GII could already be associated with the glycopeptide. To analyze this possibility, we performed IP with GII antibodies after translation of p144 and p144/97. As shown in Figure 5E, the singly glycosylated p144 did, indeed, coprecipitate with GII (lane 4, quantification in Figure 5F). Coprecipitation was independent of glucose trimming by GI, because it was unaffected by the presence of Cst (lane 3). Because Cst inhibits GII competitively, the association must have been independent of GII activity. When the glycopeptides had two glycans (p144/97), their interaction with GII was dramatically reduced (lane 12). Instead, the chains were now precipitable with α-Cnx (lane 14). However, when Cst was present during translation, significant binding of GII could be observed, and no association with Cnx was detected (lanes 11 and 13). Our interpretation of these results was that GII was recruited to arrested chains through interactions that did not involve the catalytic site in GIIα. However, this did not lead to cleavage 1. Only when a second glycan was added to the chain did cleavage 1 occur. After this trimming step, GII dissociated from the glycopeptide and was replaced by Cnx. The experiments described above were performed with truncated glycopeptides generated in microsomes. It was important to test whether the difference in trimming by GII between one and multiple glycans also applied to glycoproteins in cells. We used human pancreatic Rnase, a small well-characterized soluble protein with three glycosylation sites in Asn34, Asn76, and Asn88, as a model protein (Beintema et al., 1976Beintema J.J. Gaastra W. Scheffer A.J. Welling G.W. Carbohydrate in pancreatic ribonucleases.Eur. J. Biochem. 1976; 63: 441-448Crossref PubMed Scopus (51) Google Scholar). Because RNase does not depend on its sugars to fold, it allowed us to assess directly the importance of the number of glycans for GII trimming without interference from the quality control system. When recombinant RNase was transiently expressed in the presence of 35S-Met and immunoprecipitated with α-RNase from a cell lysate, four bands of 13, 15, 17, and 19 kDa were observed after SDS-PAGE and autoradiography (Figure 6A ). Upon treatment with PNGase F, only the fastest migrating band remained. We concluded that the four bands were variants of the RNase with no, one, two, and three glycans. The heterogeneity was due to incomplete glycosylation in all three consensus sites. We prepared lysates from pulse-labeled RNase-expressing cells chased for different times and subjected them to IP with α-RNase, α-Cnx, and α-Crt. As shown in Figure 6B (upper gel), immediately after the pulse, only RNase variants with no, one, and two glycans were detected. RNase with three glycans appeared after 4 min chase and increased over time, indicating that posttranslational glycosylation occurred. Transient, specific interaction of the newly synthesized RNase carrying two and three glycans with Cnx and Crt was clearly observed (middle and lower gels). After sequential IP with α-Cnx and α-RNase (Figure 6C), only nonspecific binding of the singly glycosylated form was detected. Quantification showed that ∼2% of the RNase with two or three glycans were associated with Cnx at 0–4 min chase, whereas none of the singly glycosylated form was specifically coprecipitated (Figure 6D). RNases with no or one glycan were secreted at least 10 min ahead of the more highly glycosylated variants (Figure 6E, quantification in Figure 6F). In the presence of Cst, secretion of the RNases with no and one glycans remained unchanged, whereas the molecules with two or three glycans were secreted 8 and 23 min faster, respectively (secretion half-times in Figure 6F). This was consistent with our previous studies showing that association with the Cnx cycle slows down glycoprotein folding and secretion (Molinari et al., 2004Molinari M. Eriksson K.K. Calanca V. Galli C. Cresswell P. Michalak M. Helenius A. Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.Mol. Cell. 2004; 13: 125-135Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The results indicated, as predicted, that whereas RNase molecules with multiple glycans were channeled through the Cnx/Crt cycle, those with no or one glycan were not. The most significant conclusion of the present work is that entry of nascent and newly synthesized glycoproteins into the Cnx/Crt cycle is regulated by the glucose trimming activity of GII. The activity is determined by at least three parameters: (1) the number of glycans present in the substrate protein (one versus more than one), (2) the location of the substrate glycoprotein (translocon associated versus released), and (3) the time of substrate exposure to GII. In contrast to GII, the trimming step catalyzed by GI is an automatic reaction that occurs immediately after glycan addition. We found that a glycan was trimmed when it was located only 72 residues from the P site, exactly the distance required for glycosylation by OST. This was consistent with a half-time of less than 2 min reported by Hubbard and Robbins, 1979Hubbard S.C. Robbins P.W. Synthesis and processing of protein-linked oligosaccharides in vivo.J. Biol. Chem. 1979; 254: 4568-4576Abstract Full Text PDF PubMed Google Scholar. The active sites of GI and OST are likely to be in close proximity to each other, and the activities of the two enzymes tightly coordinated. Unlike most glycosidases, GII is a soluble heterodimer (Trombetta et al., 1996Trombetta E.S. Simons J.F. Helenius A. Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit.J. Biol. Chem. 1996; 271: 27509-27516Crossref PubMed Scopus (200) Google Scholar). The catalytic GIIα subunit is known to retain its activity toward the chromogenic substrates when separated from the rest of the enzyme by tryptic d" @default.
• W2037439168 created "2016-06-24" @default.
• W2037439168 creator A5013589398 @default.
• W2037439168 creator A5033110590 @default.
• W2037439168 creator A5069170495 @default.
• W2037439168 date "2005-07-01" @default.
• W2037439168 modified "2023-10-11" @default.
• W2037439168 title "More Than One Glycan Is Needed for ER Glucosidase II to Allow Entry of Glycoproteins into the Calnexin/Calreticulin Cycle" @default.
• W2037439168 cites W1489462168 @default.
• W2037439168 cites W1548287977 @default.
• W2037439168 cites W1569618923 @default.
• W2037439168 cites W1584935191 @default.
• W2037439168 cites W1600323459 @default.
• W2037439168 cites W1605414441 @default.
• W2037439168 cites W1642778195 @default.
• W2037439168 cites W1946018878 @default.
• W2037439168 cites W1946381039 @default.
• W2037439168 cites W1964498671 @default.
• W2037439168 cites W1972960295 @default.
• W2037439168 cites W1985518316 @default.
• W2037439168 cites W1987216283 @default.
• W2037439168 cites W1991213853 @default.
• W2037439168 cites W1993328276 @default.
• W2037439168 cites W1997757456 @default.
• W2037439168 cites W1998957372 @default.
• W2037439168 cites W2004125187 @default.
• W2037439168 cites W2004816473 @default.
• W2037439168 cites W2004823702 @default.
• W2037439168 cites W2010770554 @default.
• W2037439168 cites W2033482754 @default.
• W2037439168 cites W2039010475 @default.
• W2037439168 cites W2039892336 @default.
• W2037439168 cites W2050479964 @default.
• W2037439168 cites W2054276303 @default.
• W2037439168 cites W2068920418 @default.
• W2037439168 cites W2076021340 @default.
• W2037439168 cites W2079230562 @default.
• W2037439168 cites W2080229382 @default.
• W2037439168 cites W2086142693 @default.
• W2037439168 cites W2087221130 @default.
• W2037439168 cites W2087662274 @default.
• W2037439168 cites W2094842946 @default.
• W2037439168 cites W212119854 @default.
• W2037439168 cites W2126753022 @default.
• W2037439168 cites W2149369491 @default.
• W2037439168 cites W2152872028 @default.
• W2037439168 cites W2153868739 @default.
• W2037439168 cites W2155368705 @default.
• W2037439168 cites W2160953366 @default.
• W2037439168 cites W978066351 @default.
• W2037439168 doi "https://doi.org/10.1016/j.molcel.2005.05.029" @default.
• W2037439168 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16039588" @default.
• W2037439168 hasPublicationYear "2005" @default.
• W2037439168 type Work @default.
• W2037439168 sameAs 2037439168 @default.
• W2037439168 citedByCount "124" @default.
• W2037439168 countsByYear W20374391682012 @default.
• W2037439168 countsByYear W20374391682013 @default.
• W2037439168 countsByYear W20374391682014 @default.
• W2037439168 countsByYear W20374391682015 @default.
• W2037439168 countsByYear W20374391682016 @default.
• W2037439168 countsByYear W20374391682017 @default.
• W2037439168 countsByYear W20374391682018 @default.
• W2037439168 countsByYear W20374391682019 @default.
• W2037439168 countsByYear W20374391682020 @default.
• W2037439168 countsByYear W20374391682021 @default.
• W2037439168 countsByYear W20374391682022 @default.
• W2037439168 crossrefType "journal-article" @default.
• W2037439168 hasAuthorship W2037439168A5013589398 @default.
• W2037439168 hasAuthorship W2037439168A5033110590 @default.
• W2037439168 hasAuthorship W2037439168A5069170495 @default.
• W2037439168 hasBestOaLocation W20374391681 @default.
• W2037439168 hasConcept C108625454 @default.
• W2037439168 hasConcept C130361206 @default.
• W2037439168 hasConcept C158617107 @default.
• W2037439168 hasConcept C206212055 @default.
• W2037439168 hasConcept C2777313579 @default.
• W2037439168 hasConcept C47450691 @default.
• W2037439168 hasConcept C55493867 @default.
• W2037439168 hasConcept C70721500 @default.
• W2037439168 hasConcept C86803240 @default.
• W2037439168 hasConcept C95444343 @default.
• W2037439168 hasConceptScore W2037439168C108625454 @default.
• W2037439168 hasConceptScore W2037439168C130361206 @default.
• W2037439168 hasConceptScore W2037439168C158617107 @default.
• W2037439168 hasConceptScore W2037439168C206212055 @default.
• W2037439168 hasConceptScore W2037439168C2777313579 @default.
• W2037439168 hasConceptScore W2037439168C47450691 @default.
• W2037439168 hasConceptScore W2037439168C55493867 @default.
• W2037439168 hasConceptScore W2037439168C70721500 @default.
• W2037439168 hasConceptScore W2037439168C86803240 @default.
• W2037439168 hasConceptScore W2037439168C95444343 @default.
• W2037439168 hasIssue "2" @default.
• W2037439168 hasLocation W20374391681 @default.
• W2037439168 hasLocation W20374391682 @default.
• W2037439168 hasOpenAccess W2037439168 @default.
• W2037439168 hasPrimaryLocation W20374391681 @default.
• W2037439168 hasRelatedWork W1497009792 @default.

• next