Monitoring of glycoprotein quality control system with a series of

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Monitoring of glycoprotein quality control system with a series of chemically synthesized homogeneous native and misfolded glycoproteins Tatsuto Kiuchi, Masayuki Izumi, Yuki Mukogawa, Arisa Shimada, Ryo Okamoto, Akira Seko, Masafumi Sakono, Yoichi Takeda, Yukishige Ito, and Yasuhiro Kajihara J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08653 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 28, 2018

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Journal of the American Chemical Society

Monitoring of glycoprotein quality control system with a series of chemically synthesized homogeneous native and misfolded glycoproteins. Tatsuto Kiuchi,† Masayuki Izumi,†# Yuki Mukogawa,† Arisa Shimada,† Ryo Okamoto,† Akira Seko,‡# Masafumi Sakono,‡# Yoichi Takeda,‡# Yukishige Ito‡§ and Yasuhiro Kajihara‡†* †Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 5600043, Japan. ‡ERATO Ito glycotrilogy project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. §Synthetic Cellular Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. ABSTRACT: The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperonetype enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon- (IFN-), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.

Introduction The glycoprotein quality control (GQC) system regulates the early stages of glycoprotein biosynthesis in the ER.1, 2 The GQC system employs many enzymes and chaperones such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (PDIs) such as ERp57, and glucosidases, for essential repetitive glycoprotein folding processes, called the CNX/CRT cycle.3, 4 (Figure. 1 A). Nascent polypeptides made by ribosomes are cotranslationally glycosylated with Glc3Man9GlcNAc2 (G3M9high-mannose type) glycans (a) at the Asn-X-Thr/Ser (X: any amino acid except for Pro) site, which start the protein folding processes. In these folding processes, the three glucoses are sequentially removed by glucosidases-I and II, and the resultant native-folded (b) and misfolded M9glycoproteins (c) (structure of M9-glycan is shown in Figure 1 B) are inspected by the folding sensor enzyme, UGGT. UGGT specifically recognizes misfolded M9glycoproteins (c) and then catalyzes the transfer of a glucose residue from UDP-glucose to the terminal of Aarm of the M9-glycan to yield G1M9-misfolded

glycoproteins (d).5-8 UGGT is responsible to find misfolded M9-glycoproteins (c) in the glycoprotein folding process, and is thus an essential folding sensor enzyme to prevent the accumulation of misfolded glycoproteins (c) in the ER. CNX and CRT are lectin chaperones that recognize the resultant G1M9-misfolded glycoproteins (d) and repair the misfolded protein structure (db) using PDIs enzymes such as ERp57, which form a complex with CNX/CRT chaperones.9 After the glycoproteins are refolded by the CNX/CRT cycle using ERp57, the glucoside is cleaved by glucosidase-II (db). These processes accumulate the resultant native-folded M9-glycoproteins (b) in the ER. Therefore, mono-glucosylation has been considered to be the emergence signal of misfolded glycoproteins (d), and CNX/CRT cycles are essential in accelerating the refolding of misfolded glycoproteins. After the folding process, native-formed glycoproteins are transported into the Golgi apparatus. Enzymes such as glycosyltransferases and glycosidases in the Golgi apparatus proceed Oglycosylation and the conversion of high mannose-type glycans into the matured acidic sialyloligosaccharides and O-glycosylation.10

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However, the individual processes of the GQC system at the molecular level are still unclear. The GQC system controls two opposite systems: the key mono-glucosylation (cd) system and de-glucosylation (db) system, using UGGT and glucosidase-II respectively, in the CNX/CRT cycle.11 Our previous studies using UGGT and UDP-glucose in simple assays showed that mono-glucosylation of misfolded glycoproteins did not result in a good yield12 and the glucosylation amount for misfolded M9-glycoproteins varied depending on the status of the misfolded form.12-14 In order to maintain an efficient refolding system, CNX/CRT needs to interact with G1M9-misfolded glycoproteins (d) before glucosidase-II action, and all misfolded glycoproteins emerging in the ER should be efficiently glucosylated. Studies with homogeneous glycoproteins probes was thus necessary to determine how the GQC cycle uses chaperones and enzymes to accelerate the quantitative refolding process.

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glucosyltransferase (UGGT) and glucosylated to accelerate refolding by lectin chaperons calnexin (CNX) and calreticulin (CRT) with protein-disulfide isomerase (PDI) or related enzymes (e.g. ERp57). This refolding system is the CNX/CRT cycle. (B) Structure of chemically synthesized homogeneous glycoproteins, erythropoietins (EPOs) bearing a homogeneous M9-high-mannose type glycan at each 24, 38 and 83 positions (3, 5, 7) and bearing three glycans 1. Misfolded EPOs bearing one M9-high mannose-type glycan at 24, 38 and 83 positions each (4, 6, 8) and bearing three glycans 2. Chemically synthesized interferon (IFN)- and interleukin (IL)-8. The blue square, green circle and red triangle are Nacetyl glucosamine, mannose and glucose, respectively.

Glycoprotein models isolated from cell expression systems or chemically prepared simple glycans have been used in the research of the GQC system, however glycoproteins bearing homogeneous M9-glycan had never been used.13, 15 Glycoproteins isolated from cell expression systems usually show considerable heterogeneity in their glycan structure. The relationship between homogeneous M9glycan structure and refolding processes in the GQC system has not been evaluated. In order to understand how the GQC system works in the ER, we have synthesized a series of homogeneous nativeand misfolded-glycoproteins such as erythropoietin (EPO), interferon (IFN)- and interleukin8 (IL-8) and used them for several biological experiments (Figure 1B). In this paper we will describe evidence that the glucosylation/deglucosylation crosstalk sustain GQC system for the acceleration of refolding processes under the fast catalytic cycle using UGGT, CNX/CRT bearing ERp57, and glucosidase-II. Results

Figure 1. Glycoprotein quality control (GQC) system and glycoproteins chemically synthesized. (A) Polypeptide synthesized by ribosome is co-translationally glycosylated by oligosaccharyltransferase (OST) and the glycosylpolypeptide bearing G3M9-glycans a starts the folding process. Misfolded glycoprotein c is recognized by the UDP-glucose:glycoprotein

To investigate the refolding processes of heavily glycosylated proteins, we chemically synthesized EPO bearing homogeneous M9-glycans. A typical mature EPO has three N-glycosyl type sialyloligosaccharides at Asn 24, 38 and 83 and an O-glycosyl type oligosaccharide at Ser126 (Figure 2A).16 A CHO expression system generates mature EPO bearing acidic sialyloligosaccharides through the Golgi. Therefore their N-glycan type is not an M9-high mannose-type, which is essential in the GQC refolding system. We synthesized three kinds of EPO bearing an M9high mannose-type glycan at 24, 38 or 83, respectively, along with EPO bearing three high mannose-type glycans at the three positions and their misfolded counterparts, resulting in eight types of EPO 1-8 (Figure 1B). As Oglycosylation of EPO is made in the Golgi apparatus, Oglycosylation at 126 serine was not essential for our research of the GQC system. For the synthesis of EPO, a corresponding homogeneous human type asparaginyl-M9-high-mannose type oligosaccharide was isolated from egg yolk and used for an improved safe Boc-solid phase peptide synthesis (SPPS).17,


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The resultant glycopeptide--thioester and peptide-thioester synthesized by SPPS were coupled by a six segment coupling strategy under native chemical ligation (NCL) conditions (Figures S12-S48).18,19 Coupling of these peptide and glycopeptide segments was performed by NCL according to our synthetic strategy as shown in Figure 2B and Figures S24-S48. Finally, folding experiments of the resultant full-length glycosyl-EPO-polypeptides were performed to yield individual native-folded structures under dialysis conditions with 6 M guanidine and redox conditions.18 Individual folding experiments yielded two kinds of products, native and misfolded forms, as shown in Figure 2C and Figures S31, S38, S43, and S48. Both products were isolated by reversed-phase HPLC and we confirmed that the sharp and broad HPLC peaks corresponded with the native-folded EPO and misfolded EPO (e.g.: Figure 2C, 5 and 6), respectively, based on mass analysis (Figure 2 D, E), disulfide bond mapping (Figures S49-56), CD (Figures S57, 58), native PAGE (Figure 2F) and in vitro proliferation activity (Figure S59, 60). The mass spectrum of misfolded EPO showed a remarkably broad signal pattern, indicating heterogeneity (Figure 2E and Figure S61). On the other hand, high-resolution mass (FT-ICR mass 9.4 Tesla) of native-form EPOs showed a gaussian-shaped mass pattern and a mass value corresponding to the desired homogeneous product (Figure 2D and Figures S31, 38, 43 and 48). These mass spectrometric analyses of individually purified EPOs were performed with direct injections of the solution into the mass spectrometer. The mass spectra were not obtained from a single time point of the LC-MS run. The misfolded EPO seemed to form aggregated forms, such as heterogeneous dimer and trimer (Figure 2F) according to native PAGE analysis. Using these processes, we synthesized homogeneous EPO bearing M9-glycans and used both native-form and misfolded EPOs for the following biological experiments. 18

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Figure 2. Synthetic strategy of erythropoietin (EPO) and their analytical data. (A) Amino acid sequence of native erythropoietin. (B) Synthetic route of EPO bearing three M9glycans by native chemical ligation and folding experiments. (C) Reversed-phase HPLC after folding experiment of EPO bearing M9-glycan at 38 position. The sharp and broad HPLC profiles correspond with native-formed EPO 5 and misfolded EPO 6. (D) High resolution ESI-mass spectrum (FT-ICR) of EPO bearing M9-glycan at 38 position 5. Calcd:20285.3948; found 20285.3562. (E) ESI-mass spectrum of misfolded EPO 6. (F) Native PAGE of native-formed EPO 5 (lane 1) and misfolded 6 (lane 2). Misfolded EPO formed an aggregated trimer.

Enzyme assay with UGGT and UDP-glucose unexpectedly showed that UGGT transferred a glucose residue to all native-form EPOs (Figure 3A) and misfolded



EPOs (Figure 3B). This result was surprising, as previously reported UGGT reactions had not shown transfer of glucose residues to any native-form glycoproteins.5, 12, 20-28 This result indicated that UGGT recognized native-form EPOs as misfolded type glycoproteins. The amount of glucosylation toward the three M9-glycans of EPO 1 was estimated by protease digestion and LC-MS analyses, and the results indicated that there was no glucosylation specificity toward the individual M9-glycans. Previously reported UGGT assays showed that UGGT definitely discriminated protein structures and transferred a glucose residue solely to misfolded glycoproteins, such as our misfolded glycosyl interleukin-8 (Figure 1B: M9IL8).5,12,20-28 Our approaches using both chemically synthesized native and misfolded M9-IL8 showed that UGGT recognized hydrophobic protein surfaces of the misfolded forms, signaling them to transfer a glucose residue onto only misfolded M9-IL8.

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type glycoproteins. We were thus able to use this nativeform EPO for the following ER lysate experiments. Next we examined how the GQC cycle works on the recognition events including native-form EPOs showing the misfolded character as well as synthetic misfolded glycoproteins in the isolated ER lysate that contains all GQC enzymes and chaperones. The ER lysate was isolated from rat liver according to the reported procedure using super centrifugation conditions.5, 32

Comparison between glycoproteins previously used and EPOs showed that previously used glycoprotein models had -sheet structure (Figure S62), where wide protein surfaces show potential hydrophilic protein surface interaction with a bulk water layer. We thought these hydrophilic surfaces might be recognized by UGGT, whereas EPO, having only helical structures that do not have wide surfaces like -sheets, could not be recognized. In order to confirm this hypothesis, we synthesized another glycoprotein, interferon (IFN)- (Figure 1B, M9IFN-) having the same helical bundle structure as EPO, and used it for confirming substrate specificity of UGGT. IFN- has the same number of amino acid residues, 166, and forms five helices structure.29 We synthesized IFN- bearing an M9-glycan at the native glycosylation position, Asn80, using a three segment coupling strategy (synthesis is shown in Figures S63-70).30 However, UGGT did not transfer a glucose residue onto this homogeneous native folded IFN- (Figure 3A, right lane). This result suggested that the hydrophilic surfaces formed on the -sheet did not stimulate UGGT activity during protein structure inspection. Based on these results, we considered the possibility that hydrophobicity of the amino acids exposed on the misfolded protein surfaces is an essential chemical characteristic in the inspection of UGGT. EPO has many hydrophobic areas exposed on the surface (Figure 4, red arrow) than that of M9-IFN-β. M9-IFN-β has also several hydrophobic areas on the surface, however these hydrophobic areas are covered by the hydrophilic amino acids (Figure 4, green arrow). Although an M9-glycan has hydrophilic properties, three M9-glycans may not sufficiently cover all of the hydrophobic surfaces on EPO. Therefore, UGGT may recognize this hydrophobicity to be a misfolded type glycoprotein characteristic. Hydrophobicity31 of both EPOs and M9-IFN-β are also discussed in Figures S1-3. Although the interaction of the EPO isoforms with UGGT was an unexpected finding, this property allowed us to obtain stable monomeric misfolded

Figure 3. UGGT glucosyltransfer toward synthetic glycoproteins by isolated UGGT enzyme. (A and B) Amount of glucosyltransfer toward native-formed and misfolded EPO by isolated UGGT. Blue, red and light green are 1, 2, and 3 h reaction, respectively. Quantity of M9-EPO and G1M9-EPO were estimated by the intensity of ESI-mass peak. Amount of EPO substrate used was 50 pmol.

However, eight EPOs of native and misfolded form were not efficiently glucosylated by UGGT in the isolated ER lysate (Figure 5A:native form and Figure S4A:misfolded form). UGGT prepared by cell expression clearly recognized the EPO to be the misfolded type hydrophobic character, and transferred a glucose to each native-folded and misfolded EPO (Figure 3). We considered the possibility that glucosidase-II cleaves the G1M9-glycan immediately after glucosylation by UGGT, resulting in poor accumulation of G1M9-glycan in the ER lysate. Several groups discussed the existence of


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shuttle type glucosylation/deglucosylation processes in the ER.1,11,33 The Kearse group successfully monitored glucosylation/deglucosylation processes by the chased method. However based on those studies, the relationship between refolding processes and amount of glucosylation was unclear. Therefore, we examined glucosylation reactions of both native-form EPO and misfolded EPO in the ER lysate assay in the presence of a potent glucosidase II inhibitor, deoxynojirimycin (DNJ) (Figure 5B and Figure S4B). The assays clearly showed that glucosylation by UGGT remarkably increased to 30-40 % toward EPOs bearing one and three M9-glycans (Figure 5B and Figure S4B). However, we could not observe high-yield glucosylation for all glycoprotein probes.

place between the M9-glycan and G1M9-glycan, likely in a crosstalk manner that we could not observe any glucosylation. Next we investigated the refolding ability of ER lysate toward misfolded synthetic glycoproteins during the fast glucosylation/deglucosylation-crosstalk. However, glycosylpolypeptide of EPOs didn’t show suitable solubility in the ER lysate due to their hydrophobic nature. Because we had already synthesized homogeneous monomisfolded and dimeric-misfolded M9-IL8 by scrambling the disulfide bonds,12 we used it for the evaluation of refolding activity in the ER lysate. As shown in Figure 6A and B, the two HPLC profiles show transitions from misfolded monomeric (Figure 6A) and dimeric (Figure 6B) glycoprotein forms to native-folded M9-IL8. Comparison to our authentic native-folded and misfolded M9-IL812 determined that the product in the ER lysate was nativefolded M9-IL8, which was confirmed using the LC-MS monitoring system. We also confirmed that heatdenatured ER lysate (reflux for 5 min) did not refold M9IL8. These results indicated that isolated ER lysate had almost all active enzymes and chaperones necessary in the GQC system. However, the reactions did not show high yield glucosylation during refolding processes.

Figure 4. UGGT recognizes hydrophobic surfaces exposed on the protein surface. Hydrophobic (yellow) and hydrophilic (blue) surface of EPO and IFN-.

In terms of this deglucosylation process, we needed to confirm whether G1M9-EPO yielded M9-EPO rather than G1M8-EPO which was generated by ER-mannosidase. In order to confirm this process, we added UDP-[1-13C]glucose into ER lysate assays. ER-lysate assay by highresolution mass analysis showed that [1-13C]-G1-M9-EPO was accumulated in the presence of DNJ, and that M9-EPO was generated rather than [1-13C]-G1-M8-EPO in the absence of DNJ (Figures S5, S6). These results strongly indicated that the fast glucosylation/deglucosylation took

Figure 5. UGGT glucosyltransfer toward synthetic glycoproteins by isolated ER lysate. (A) Glucosylation of synthetic EPO in ER lysate. Quantity of M9-EPO and G1M9EPO were estimated by the intensity of ESI-mass peak. Total amount of EPO used in the individual assay was 50 pmol. Because EPO 1 has three M9-glycans, total glucosylation is high. (B) Glucosylation of synthetic EPOs in ER lysate in the presence of potent glucosidase inhibitor, 1-deoxynojirimycin (DNJ). Quantity of M9-EPO and G1M9-EPO were estimated



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by the intensity of ESI mass peak. Total amount of EPO used in the individual assay was 50 pmol.

We also examined refolding in the ER lysate for the misfolded EPO that was an aggregated form having an M9glycan at 38 position. Figure 6C shows the refolding process monitored by LCMS, and it indicates that less than 20% of misfolded EPO was refolded. There was also a small amount of glucosylation toward the misfolded EPO in the ER lysate (Figure S4A). Even potential chemical denaturation treatment with 6 M Gn-HCl and tris(2carboxyethyl)phosphine (TCEP) toward the misfolded EPO did not give linear glycosyl-EPO peptide in good yield (Figure S61). These results indicated that misfolded glycoproteins might have complex structure and thus difficult to return to their linear or native forms. These heavily misfolded glycoproteins may leave from the GQC cycle, and then be transported to the systems for metabolizing such as the ERAD process.1

Figure 6. Refolding of misfolded glycoproteins in the ER lysate. (A) Refolding of monomeric misfolded glycosyl IL-8 bearing disulfide bonds (Cys9-34 and 7-50) to native form glycosyl IL8 bearing disulfide bonds (Cys 7-34 and 9-50). (B) Refolding of dimeric misfolded glycosyl IL-8 bearing disulfide bonds (Cys7/9-7/9 and 34-50) to native form glycosyl IL8 bearing disulfide bonds (Cys 7-34 and 9-50). (C) Refolding of misfolded EPO having a glycan at 38. Native form EPO was barely observed by ESI mass signals as 13+, 14+ and 15+ ion peaks. Asterisks * indicate impurities.


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Journal of the American Chemical Society shown in (B). The bottom three mass spectra were observed at 6.0 min retention time shown in (B). Blue mass values and dotted line indicate mass value of mono-glucosylated G1M9IL8s. (D) Monitoring of refolding by base peak chromatography in the absent of glucosidase-II inhibitor (DNJ). Reaction conditions and monitoring system were similar to that of experiments shown in B and C. Peaks observed around 7-9 min are unknown ER proteins. (E) Monitoring of refolding by LC-MS in the absent of glucosidase-II inhibitor (DNJ). The top four mass spectra were observed at 6.6 min retention time shown in (D). The bottom three mass spectra were observed at 6.0 min retention time shown in (D). Blue mass values and blue dotted line correspond with mass value of mono-glucosylated G1M9-IL8s. Red mass values and red dotted line correspond with native M9-IL8s. Misfolded M9-IL8 was not observed at 6.6 retention time. Asterisks * indicate impurities.

We then examined the relationship between the amount of glucosylation and refolding process in the ER lysate. Figures S7-9 show the monitoring of the refolding processes of misfolded M9-IL8 and its dimer by LC/MS. Refolding processes were clearly observed, but efficient glucosylation was not observed for misfolded M9-IL8 and its misfolded dimer form. Both misfolded forms showed less than 30 % glucosylation (Figures S7C and S9C) and there was no glucosylation toward the refolded native form. However, the same refolding assay in the presence of DNJ increased the amount of glucosylation toward the refolded native form to 30 % yield. These results indicated that the GQC system yielded misfolded G1M9glycoproteins or G1M9-folding intermediates, and the resultant G1M9 glycan of the native-glycoproteins remained because of the inhibition of glucosidase-II. The refolding efficiently proceeded, because DNJ specifically inhibited glucosidase-II and did not inhibit the refolding system of CNX/CRT bearing ERp57. However the glucosylation yield was still low.

Figure 7. Relationship between glucosylation/deglucosylation and refolding ability of misfolded G1M9IL8 in the ER lysate. (A) Refolding of homogeneous misfolded G1M9-IL8 to native M9-IL8 in the ER lysate. Synthesis of homogeneous G1M9-IL8 was previously reported (13). (B) Monitoring of refolding by base peak chromatography in the presence of glucosidase-II inhibitor (DNJ). The peak observed at 6.6 min and 6.0 min were misfolded G1M9-IL8 and native G1M9-IL8, respectively. Peaks observed around 7-9 min are unknown ER proteins. (C) Monitoring of refolding by LCMS in the presence of glucosidase-II inhibitor (DNJ). The top four mass spectra were observed at 6.6 min retention time

In order to confirm whether GQC can regulate an efficient refolding during the fast glucosylation/deglucosylation process, we needed to confirm that G1M9-misfolded glycoproteins formed once could be quantitatively converted into native M9glycoproteins. In order to perform this assay, homogeneous misfolded-G1M9-glycoproteins as a starting substrate were essential to evaluate the process in detail. UGGT did not give G1M9-glycoprotein in high yield, and purification of G1M9-glycoprotein from M9-glycoprotein by HPLC was very difficult due to overlap on HPLC chromatography. However, recently we were able to synthesize a homogeneous misfolded monomeric-G1M9IL8 by a unique chemoenzymatic-route from misfolded dimeric-G1M9-IL8 through refolding processes.13 We used homogeneous misfolded monomeric-G1M9-IL8 thus synthesized to confirm whether a homogeneous misfolded monomeric- G1M9- IL8 was recognized by the GQC system and converted into native M9-IL8 in quantitative yield. The refolding experiments were performed with a homogeneous misfolded G1M9-IL8 in either the presence



or absence of DNJ in the ER lysate (Figure 7 and Figure S10). Figure 7 clearly shows that misfolded G1M9-IL8 was efficiently converted into native M9-IL8 or native G1M9IL8 in the absence or presence of DNJ, respectively. Figure S10 also shows the same process of misfolded dimer in the present of DNJ. These data indicated that glucosidase-II reactions during and after the refolding processes were efficiently inhibited by DNJ. Our experiments using homogeneous glycoproteins clearly demonstrated that the GQC system has the ability to perform quantitative refolding of misfolded G1M9-glycoproteins to native M9glycoproteins. Discussion The folding of glycoproteins is likely regulated by the entire GQC system. Many chaperones and enzymes exist in the ER and their functions are to accelerate folding of proteins, including glycoproteins, and these molecules may cooperate with each other to achieve their function. Glycoproteins have diverse forms, such as multi domains, and can vary in size. Misfolded glycoproteins may have more complex and diverse structures. If UGGT, CNX/CRT, and glucosidase-II form a fixed complex form, the GQC system may be unable to refold such diverse patterns of misfolded glycoproteins. The Parodi group reported that UGGT, CRT/CNX and glucosidase-II do not form an efficient complex.11 Our group also could not confirm the existence of a fixed complex form by the use of a cross linker reagent (data not shown). We thus hypothesized that UGGT, CRT/CNX and glucosidase-II act independently, rather than forming a complex. In our previous experiments, glucosidase-II reaction toward both misfolded and native G1M9-glycoproteins were inhibited by the addition of CRT.13 These results indicated that CRT and glucosidase-II might not form a complex to cleave G1M9-glycan and accelerate refolding processes. In these cases, UGGT may form a complex with Sep15 enzyme using seleno-cysteine for the reduction of undesired disulfide bonds.11, 34 Based on our data, we hypothesize that the GQC cycle employs a catalytic cycle for the acceleration of refolding processes. The Parodi group suggested the existence of shuttle processes between glucosylation and deglucosylation processes. Our data suggested that glucosylation and deglucosylation processes were very fast processes that could not be detected by LC/MS, indicating that the refolding of misfolded glycoproteins occurred during the fast glucosylation/deglucosylation process. Although refolding processes by the GQC cycle did not give high glucosylation yield, we clearly observed quantitative refolding processes from homogeneous G1M9-misfolded glycoprotein to native M9-glycoprotein (Figure 7). These observations indicated that the refolding process of the GQC system employing fast glucosylation/deglucosylation seemed to be a catalytic refolding cycle. Therefore, UGGT glucosylation seemed not to be highly active, although a considerable amount of UGGT had been found in the ER as an ER specific

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enzyme.20 In our other experiments using a small glycoprotein, refolding was clearly inhibited by anti CNX or CRT polyclonal antibody (data not shown). Although the research is still in progress, these results indicated refolding of misfolded glycoproteins to be dominated by the GQC system. We hypothesized that the GQC system works in one of two modes: an ordered cycle mode or a random by chance mode. The Totani and Ito group reported that G1M9glycan still interacts with UGGT after UGGT inspection/glucosylation and proved that the addition of CRT accelerates the dissociation of the G1M9-glycan and UGGT complex35. The reported Km value of UGGT for misfolded M9-glycoprotein and CNX/CRT for G1M9glycoprotein was in the submicro-micro molar range,14, 36, 37 while glucosidase-II shows a larger Km value.38 Based on these kinetic results, the GQC cycle appears to be an ordered cycle consisting of UGGT, CNX/CRT-ERp57, and then glucosidase II. Misfolded glycoproteins synthesized by the scrambling of disulfide bonds could be refolded in the ER lysate (Figures 6A, B). However, the synthetic misfolded EPOs showed very poor refolding in the ER lysate (Figure 6C). Our experiments also showed that misfolded EPO were found to form an aggregated structure (Figure 2E, F), which may have been difficult to undergo refolding in the ER. Indeed, even potential chemical denaturation with TCEP and 6M guanidine did not give a linearized glycosyl polypeptide from the misfolded EPO in good yield (Figure S61). Accumulation of such misfolded glycoproteins can result in dire effects within an organism, and these misfolded glycoproteins are metabolized by ER associated degradation system.1 Our laboratory has also studied the interaction of UGGT with small hydrophobic M9-glycopepetide by 1H-15NHSQC NMR experiments.39 These experiments indicated that UGGT could interact with specific hydrophobic amino acids of small hydrophobic M9-glycopepetides. Our experiments described here also indicated that native EPOs having hydrophobic protein surface were glucosylated by UGGT. A similar case was reported by the Caramelo group.40 They reported that UGGT transferred a glucose to a correctly folded monomeric soybean agglutinin (SBA), while UGGT did not to transfer a glucose to natural form SBA that is a tetramer. The Caramelo group concluded that UGGT recognized the hydrophobic interfaces which are hidden by other subunits in the structure of native tetramer. In addition to these experiments, structural comparison between EPO and IFN- (Figure 4) enabled us to discuss what sort of hydrophobic surface was recognized by UGGT. These results turn out that UGGT is prone to recognize hydrophobic surfaces exposed on the protein surface and may not be able to recognize whether those are a native structures or misfolded forms. Recently the two research groups have reported the structure of UGGT.7,8 The UGGT indeed has unique multiple domains which can cope with diverse misfolded pattern for the identification of misfolded glycoproteins. Our experimental results give


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additional insight into recognition system of UGGT and GQC. In conclusion, we successfully synthesized a series of homogeneous glycoproteins bearing M9-high mannosetype glycans such as four kinds of EPO, IFN-, IL8, and their misfolded forms including G1M9-IL8. In terms of the misfolded forms, we were able to prepare several unique protein structures such as monomeric misfolded forms, dimeric misfolded forms, and aggregated forms. Experiments using all of these probes suggested that the GQC system act in a catalytic ordered cycle to accelerate the quantitative refolding of misfolded glycoproteins and this system was organized by fast glucosylation/deglucosylation crosstalk. Our experiments also provide clear evidence that a singular glucose residue is a critical emergence signal in the formation of misfolded glycoproteins in the GQC system. We examined with ER lysate, and the chemically synthesized homogeneous glycoprotein models showed clear molecular recognition within the GQC system.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. All experimental procedures, characterization, NMR spectra, and MS spectra (file type, PDF).

AUTHOR INFORMATION Corresponding Author [email protected]

ORCID Masayuki Izumi: 0000-0001-6486-9678 Ryo Okamoto: 0000-0001-9529-2525 Yukishige Ito: 0000-0001-6251-7249 Yasuhiro Kajihara: 0000-0002-6656-2394

Present Addresses Prof. Dr. Masayuki Izumi: Department of Chemistry and Biotechnology, Faculty of Science and Technology, Kochi University, 2-5-1 Akebonocho, Kochi, Kochi 780-8520, Japan., E-mail: [email protected]; Dr. Akira Seko: Japan Agency for medical Research and Development, 1-7-1-22, Otemachi, Chiyodaku, Tokyo, 100-0004. Japan., E-mail: [email protected]; Associate Prof. Dr. Masafumi Sakono: Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku,Toyama 930-8555, Japan, E-mail: [email protected]; Associate Prof. Dr. Yoichi Takeda: Department of Biotechnology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan, E-mail: [email protected]. Dr. Yasuhiro Kajihara:Project Research Center for Fundamental Science. Funding Sources

This work was also supported by financial support from the Japan Society for the Promotion of Science (KAKENHI Grant Number 16H06290 to Y.I.).

ABBREVIATIONS GQC:glycoprotein quality control; ER:endoplasmic reticulum; UGGT:UDP-glucose:glycoprotein glucosyltransferase; CNX:calnexin; CRT:calreticulin; PDI: protein disulfide bond isomerase; EPO: erythropoietin; IFN- interferon-; IL8:interleukin; SPPS: solid phase peptide synthesis; NCL:native chemical ligation; DNJ: deoxynojirimycin.

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Monitoring of glycoprotein quality control system with a series of

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