Bifunctionality of Glycan-Recognizing Proteins in N-Glycoprotein Biosynthesis Taiki Kuribara and Kiichiro Totani*

Totani K. et al. Medical Research Archives, vol. 6, issue 9, September 2018 Page 1 of 14

REVIEW ARTICLE

Bifunctionality of glycan-recognizing proteins in N-glycoprotein biosynthesis Taiki Kuribara and Kiichiro Totani*

Authors’ affiliations:

Department of Materials and Life Science, Seikei University 3-3-1 Kichijoji-kitamachi, Musashino, Tokyo 180-8633, Japan

*Correspondence: Kiichiro Totani, Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino, Tokyo 180-8633, Japan Tel.: +81-422-37-3745; Fax: +81-422-37-3871; E-mail: [email protected]

Abstract

In an early step of N-glycoprotein biosynthesis, high-mannose type glycans are attached to nascent polypeptides in the endoplasmic reticulum. Subsequently, the glycopolypeptides adopt the correct folding conformations, after which glycans are converted to complex type glycans in the Golgi apparatus. During these processes, over 30% of nascent glycoproteins exist in either misfolded or unfolded forms, therefore proper folding of these proteins is essential for efficient N-glycoprotein biosynthesis. Furthermore, elimination of misfolded glycoproteins from the endoplasmic reticulum via cytoplasmic degradation is essential for maintenance of cellular homeostasis. Through these sorting processes, various glycan-recognizing proteins, such as glycosyltransferases, glycosidases, and lectins, contribute to the conversion of nascent glycoproteins into a variety of glycoforms. Some glycans are believed to be signals for the folding, secretion, and degradation of glycoproteins that are partially folded, correctly folded, and misfolded, respectively. Since targets of the glycan-recognizing proteins are diverse within the body, some of the lectin and glycan processing enzymes may be bifunctional, characterised by dual recognition of both glycan and aglycone moieties. In fact, uridine 5’-diphosphate glucose: glycoprotein glucosyltransferase, which localises in the endoplasmic reticulum, has been known to have both, glucose transfer activity and the ability to sense improperly folded proteins. Similar dual recognition properties have been reported, particularly in glycan-recognizing proteins, with regards to glycoprotein folding, sorting, and degradation processes. However, there have not been any review articles highlighting the bifunctionality of glycan-recognizing proteins thus far.

In this review, we summarise several examples of the bifunctionality of glycan-recognizing proteins involved in N-glycoprotein biosynthesis.

Keywords: N-glycoprotein biosynthesis, glycan-recognizing protein, aglycone recognition, bifunctionality

1. Introduction glycan conversion likely depends on both the glycan structure and the folding state of In N-glycoprotein biosynthesis, various the protein. In fact, activity of uridine glycan-recognizing proteins contribute to 5’-diphosphate (UDP)-glucose: glyco- glycan processing, as well as protein-folding protein glucosyltransferase (UGGT) has and degradation. The N-glycoprotein bio- been known to differ between native, mis- synthesis processes are categorised as fol- folded, and unfolded monoglucosylated lows: 1) High- mannose-type gly- glycoproteins.8 A similar phenomenon has can-processing, glycoprotein folding, and been reported for several other glycan sorting in endoplasmic reticulum (ER), 2) processing enzymes and lectins. In this re- Structural conversion of folded glycoprote- view, we will provide an outline on the bi- ins, such as the conversion of functionality (the simultaneous dual recog- high-mannose-type glycans to complex-type nition of glycan and protein moiety) of glycans, in the Golgi apparatus, and 3) glycan-recognizing proteins in N-glyco- Glycan removal and degradation of mis- 1-7 protein biosynthesis. A summary of the bi- folded glycoproteins in cytosol (Figure 1). functionality of typical glycan recognizing Since the targets of glycan- recognizing proteins highlighted in this review is listed proteins are extremely diverse within the in Table 1. body, glycan recognition and the related

2. Glycoprotein folding and sorting in the In this section, we focus on the bifunctio- ER nality of ER enzymes and lectins during signalling pathways that involve glyco- Within the ER, glycan structures on glycoproteins. proteins play an essential role for the secretion and degradation of glycoproteins.1-6 In 2.1. Malectin terms of determining signal glycans, the folding state of the glycoprotein could affect In 2008, Schallus et al. reported a novel glycan-related enzymatic or lectin activities. lectin named malectin that is involved in the

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Totani K. et al. Medical Research Archives, vol. 6, issue 9, September 2018 Page 4 of 14 early steps of N-glycoprotein biosynthesis in monoglucosylated glycoproteins in the ER.9 Malectin recognises ER.14,15,16 CNX binds to the folding inter-

Glc2Man9GlcNAc2 (G2M9), which is pro- mediate of class I histocompatibility mole- duced at the initial step of ER glycoprotein cules,16 T cell receptors, membrane immu- processing. The target epitope of malectin noglobulins,17 influenza hemagglutinin,18 was revealed to be the Glc1-3Glc moiety and transferrin.19 As the protein nears its based on frontal affinity chromatography10,11 correct folded conformation, the glyco- and crystallographic studies.9,12 Thus, the proteins dissociate from CNX. CRT pos- lectin selectively binds diglucosylated high sesses a similar dissociation mode for mo- mannose N-glycan. Several studies have noglucosyated glycoproteins.20,21 Taken proposed the bifunctionality of malectin. In together, CNX/CRT bind to partially folded brief, the lectin associates with the mis- glycoproteins as part of a glycoprotein folded 1-antitrypsin null Hong Kong va- quality control system. Inversely, Helenius riant rather than the wild-type et al. reported that only monoglucosylated 1-antitrypsin,12 indicating that malectin N-glycan is required for CRT in vitro.22 Thus, selectively associates with a model substrate whether CNX/CRT have bifunctionality for ER-associated degradation (ERAD). towards glycoproteins remains controversial. Furthermore, overexpression of malectin We focused on the bifunctionality of CRT, enhances ERAD.12 Although it has been and carried out an interaction analysis using shown that malectin forms a complex with a series of structurally-defined ribophorin I to recognise misfolded pro- G1M9-derivatives with different aglycone teins,13 immunoprecipitation studies high- moieties on proximal regions at light that malectin may directly bind to N-glycosylation sites (-OH, -Gly, and misfolded ERAD substrates, suggesting the -Gly-Glu-tBu).23 Binding assays between bifunctionality of the lectin.12 Although the CRT and these substrates revealed diverse precise mechanism of malectin bifunctio- specificity. CRT bound strongly to nality requires further investigation, this G1M9-Gly-Glu-tBu, but showed moderate activity likely serves to prevent aggregation binding to G1M9-Gly. Moreover, weak in early N-glycoprotein biosynthesis via binding relative to these derivatives was detection of nascent unfolded glycoproteins. observed when using G1M9-OH as a ligand. These results indicate that CRT simultaneously recognises the G1M9-moiety and 2.2. Calnexin and Calreticulin aglycone structures at regions close to Removal of the outermost glucose-residue N-glycosylation sites depending on their of the G2M9-protein produces hydrophobicity. In other words, CRT dis-

Glc1Man9GlcNAc2 (G1M9)-protein, which plays bifunctionality when recognising is recognised by calnexin (CNX) / calreti- G1M9-derivatives. In order to better under- culin (CRT) to accelerate protein folding. stand this bifunctionality, we examined the CNX and its soluble homologue CRT are interaction of CRT with a series of lectin-like molecular chaperones that bind to G1M9-derivatives exposed to hydrophobic

BODIPY-dye connected with hydrophilic vitro simultaneous monitoring of the reglu- ethylene glycol linkers of varying lengths cosylation and folding process using che- (n = 0, 4, 8, and 12).24 These experiments moenzymatic synthesised crambin as a revealed that CRT preferentially binds to model substrate-glycoprotein.33 Their find- G1M9-BODIPY with a shorter linker ings indicate that UGGT can recognise each

(binding orders; EG0 > EG4 > EG8 > EG12), of the folding-intermediates during the highlighting that CRT binding depends on folding process. Sousa and Parodi reported the hydrophobicity of the regions close to that UGGT recognises hydrophobic non- the N-glycosylation site. apeptides more than hydrophilic ones.26 On the other hand, Totani et al. previously synthesised non-peptidic substrates of UGGT, 2.3. UGGT i.e. thirteen structurally-defined M9-glycans After G1M9-protein undergoes a folding conjugated with various hydrophobic agly- process mediated by CNX/CRT, the glucose cones.34 The reactivity of UGGT towards residue is removed by glucosidase II to these synthetic substrates and the recogni- 25 produce Man9GlcNAc2 (M9)-proteins. tion motif of the enzyme were clearly iden- The resulting M9-protein is recognised by tified, thus contributing to our understanding the folding sensor enzyme UGGT.8,26 UGGT of the molecular basis behind the bifunc- specifically glucosylates the tionality of UGGT. Interestingly, reactivity M9-glycoprotein, resulting in a molten orders of M9-Gly-Glu-tBu, M9-Gly, and globule-like folding intermediate state. M9-OH to UGGT were similar to that of Conversely, this enzyme displayed little G1M9-derivatives to CRT.23,34 These results recognition for properly folded and severely indicate that UGGT and CRT recognise misfolded random-coil-like M9-protein.8 A these glycoprobes in a similar bifunctional unique specificity has been observed for not manner. Moreover, binding of synthetic only natural glycoproteins, but also syn- M9-derivative changed the circular dich- thetic glycoprobes, such as denatured gly- roism (CD) spectrum of UGGT only when coproteins,27,28 neoglycopeptides,29 syn- M9-glycan was conjugated with hydro- thetic homogeneous glycoproteins,30 and phobic aglycone (5-TAMRA).35 This change structurally-defined synthetic substrates.31 in the CD spectrum strongly suggests that To obtain a precise understanding of target UGGT simultaneously recognises recognition via UGGT, Trombetta et al. M9-glycan and hydrophobic aglycone. Ha- reported that chemically denatured ribo- chisu et al. reported UGGT has flexible nuclease B (RNaseB) was strongly gluco- recognition mode for pseudo-misfolded sylated by the enzyme, while native RNaseB glycoproteins.36 The authors compared the was only slightly reglucosylated.32 Fur- glucosyltransfer activity of UGGT towards thermore, no UGGT activity was reported M9-methotrexate complexes with hydro- for the fully unfolded RNaseB. These in phobic pyrene-labelled dihydrofolate re- vitro studies clearly showed the bifunctio- ductase (DHFR), which modifies cysteine nality of UGGT. Kajihara et al. reported in groups at different positions of DHFR by

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Totani K. et al. Medical Research Archives, vol. 6, issue 9, September 2018 Page 6 of 14 pyrene groups. According to these results, related to glycoprotein sorting,

UGGT displays flexibility for sensing hy- Man8BGlcNAc2 (M8B) and drophobic surface patches. In fact, Satoh et Man8A/CGlcNAc2 (M8A and M8C) that are al. recently reported that UGGT possesses a produced by -1,2-mannosidases in the ER small catalytic domain and a flexible rec- are believed to be secretion and degradation ognition domain for hydrophobic patches.37 signals, respectively.44 However, it remains This attribute likely allows UGGT to sense a unclear which of the -1,2-mannosidases, variety of glycoprotein folding interme- particularly among the EDEM family, pro- diates. Therefore, the flexible recognition of duce glycoprotein secretion (M8B) and surface hydrophobicity is important for the degradation (M8A and M8C) signals. This is bifunctionality of UGGT. Focusing on the likely because EDEM activity has not yet proximal regions of N-glycosylation sites, been detected in vitro. Therefore, the activity we synthesised a series of chitobi- of these mannosidases has been inferred ose-pentapeptides as inhibitors of UGGT.38 using transgenic cell lines (i.e. overexpres- Six pentapeptides with systematically con- sion or knockout of EDEMs).43,45,46 Ac- trolled hydrophobic leucine and hydrophilic cording to these studies, all EDEMs possess serine sequences were synthesised. With mannosidase activity within intact cells. In these six pentapetides, UGGT inhibition brief, it is known that EDEM1 trims the A assays were carried out. UGGT specifically and C branch, EDEM2 trims the B branch, recognised the serine residue directly linked whereas the branch specificity of EDEM3 to the C-terminus of N-glycosyl-asparagine. has not yet been identified. Despite various Taken together, these findings increase our efforts towards analysing the specificity of understanding of the molecular basis behind -1,2-mannosidases, it is still unclear the bifunctionality of UGGT. whether these glycosignals are produced randomly or regioselectively. Recently, we succeeded in selectively manipulating dis- 2.4 -1,2-Mannosidases in the ER crete -1,2-mannosidase activities using Through a glycoprotein sorting step in the reciprocally selective inhibitors against ER, mannose residues of M9-glycoproteins glycosignal production.47 The results sug- are sequentially trimmed by -1,2-manno- gested that secretion and degradation gly- sidases. Tri-branched M9-glycan contains cosignals are regioselectively produced by cleavable -1,2-mannoside linkages in the discrete -1,2-mannosidase(s), and that two outermost sugar-residue of each branch. independent pathways for glycosignal pro- Several reports suggest that four mannosi- duction exist in the ER. Very recently, Ho- dases are responsible for trimming sokawa and co-workers reported that in vitro -1,2-mannosides in the ER: ER mannosi- EDEM3 activity requires covalent interac- dase I (ERManI),39,40 ER-degradation- en- tion with ER-resident protein 46.48 Fur- hancing -1,2-mannosidase-like protein 1 thermore, Hebert et al. reported that the (EDEM1),41 EDEM2,42 and EDEM3.43 mannosidase-like domain of EDEM1 pos- Based on the lectin/enzyme specificities sesses mannosidase activity by using a ER

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Totani K. et al. Medical Research Archives, vol. 6, issue 9, September 2018 Page 7 of 14 mannosidase I/Golgi mannosidase IA/B/C terms of redox conditions in the ER, quadruple knockout cell.49 Therefore, the in EDEM1 showed additional bifunctionality, vitro mannosidase activity of the EDEM such as covalent-like disulfide bond forma- family has been partially understood, al- tion between cysteine residues of EDEM1 though in vitro branch specificity is still and the target protein.49 under investigation. With regards to aglycone structure recognition by -1,2-manno- 2.5. OS-9 sidases, several studies indicate that ER- ManI, EDEM1, EDEM2, and EDEM3 are The action of ER -1,2-mannosidases pro- able to recognise aglycone structures. ER- vide several glycoforms of high mannose ManI preferentially trims denatured glycans. Particularly, the glycoforms lacking M9-bovine thyroglobulin and soybean ag- an outermost mannose residue at the glutinin.50 The enzyme can recognise the C-branch are believed to serve as degrada- polypeptide region of substrate M9-proteins, tion signals.44 Proteins with these glycosig- thus suggesting the bifunctional nature of nals are captured by osteosarcoma 9 (OS-9) ERManI. EDEM1 and EDEM2 also have located at a dislocon channel, which aids in been reported to bind to hydrophobic sur- delivering misfolded glycoproteins from the faces of model misfolded proteins.51 Thus, ER to the cytosol.52,53 Based on its role in EDEM1 and EDEM2 seem to have bifunc- facilitating degradation of misfolded glyco- tionality towards misfolded glycoproteins. proteins, OS-9 might possess discrimination In addition, putative ERAD target proteins ability based on hydrophobicity. In fact, have been reported to bind to the hydro- OS-9 preferentially binds to a fold- phobic surface patch in EDEM1’s manno- ing-defective, glycoprotein-null, Hong sidase-like domain.49 In any case, EDEM1 Kong variant of 1-antitrypsin.53 It has also and EDEM2 display bifunctionality via their been reported that OS-9 forms a complex glycan-binding ability and ability to discri- with glucose-regulated protein 94, which minate between different hydrophobicities. functions as a molecular chaperone in the In addition, we examined mannose trimming ER.54 Although several findings suggest the of M9-glycan linked to BODIPY-dye with a bifunctionality of OS-9, further investiga- systematic series of linker lengths in the ER tion will be necessary for precise under- of mouse liver samples.24 A higher mannose standing of its molecular basis. trimming velocity was observed for the substrates with shorter linkers. Since mul- 3. Glycoprotein degradation in cytosol tiple -1,2-mannosidases were tested in these experiments, it remains unclear which Retrotranslocated misfolded glycoproteins mannosidase(s) is responsible for the man- are deglycosylated in the cytosol, followed nose-trimmings. However, at least a sin- by proteasomal degradation.55 A deglyco- gle-1,2-mannosidase might discriminate sylation reaction likely contributes to effi- hydrophobic surfaces on the proximal region cient degradation of misfolded proteins.56 In at N-glycosylation sites. Interestingly, in light of the fact that deglycosylation is ne-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Totani K. et al. Medical Research Archives, vol. 6, issue 9, September 2018 Page 8 of 14 cessary for misfolded glycoprotein degra- bifunctionality could be conserved in dation, we focused on the bifunctionality of mammalian cytosolic PNGase. cytosolic deglycosylation enzyme. 4. Future perspectives 3.1. PNGase In this review, we focused on the bifunc- Peptide: N-glycanase (PNGase) is a type of tionality of N-glycoprotein biosynthesis. amidase that cleaves amide bonds between Several lectins and enzymes possess the N-glycan and asparagine.57 Although vari- ability to recognise not only glycans, but ous types of PNGase have been identified in also aglycones (hydrophobicity and/or pep- nature,58-61 biological significance has only tide sequence). In the early steps of been linked to cytosolic PNGase, which N-glycoprotein biosynthesis in the ER, plays an essential role in misfolded glyco- newly synthesised G2M9-polypeptides are protein degradation.57 Glycan specificity of recognised by malectin. Malectin likely cytosolic PNGase has been reported,62 al- possesses bifunctionality towards G2M9- though the related peptide specificity has not polypeptides to prevent aggregation of nas- been precisely understood. We therefore cent misfolded G2M9-polypeptides. After analysed the peptide specificity of PNGase removal of the outermost glucoside linkage towards chitobiose-pentapeptides using of G2M9-protein, the resulting systematically controlled hydrophobic leu- G1M9-protein is recognised by CNX and its cine and hydrophilic serine sequences as soluble homologue CRT. In this step, these substrates.63 Yeast cytosolic PNGase chaperones simultaneously recognise gly- showed not only broad capacity for these can- and aglycone-moieties. The bifunctio- substrates, but also preferential deglycosy- nality contributes to accelerated protein lation for chitobiose-pentapetides with pep- folding and delivery to glucosidase II for tide sequences that were more hydrophobic. further glucose cleaving. The resulting Namely, PNGase would discriminate be- M9-protein is then recognised by the en- tween both glycan-moieties and peptide zyme UGGT, which possesses glucose sequences. Interestingly, subsequent kinetic transfer activity and folding sensor ability. analysis revealed that cytosolic PNGase may The bifunctionality allows for the monitor- act not only as a Km dependent enzyme for ing of folding states and the reglucosylation some substrates, but also as a Vmax depen- of misfolded M9-protein to provide dent enzyme for other substrates. The high G1M9-protein to maximise folding with catalytic range of yeast cytosolic PNGase CNX/CRT. After sensing the folding status may be one of the reasons for the broad of M9-protein, -1,2-mannosidases in the capacity toward a variety of misfolded gly- ER trim the mannose-residues to produce coproteins. This seems to be a kind of glycosignals for secretion and degradation. another bifunctionality. A similar glyco- During these steps, the bifunctionality of protein quality control machinery has also -1,2-mannosidases might be useful for been reported in yeast.64 Therefore, similar discriminating properly folded and mis-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Totani K. et al. Medical Research Archives, vol. 6, issue 9, September 2018 Page 9 of 14 folded glycoproteins. During downstream References N-glycoprotein biosynthesis in the ER, 1. Caramelo JJ, Parodi AJ. A sweet code misfolded glycoproteins must be properly for glycoprotein folding. FEBS Lett. transferred to degradation pathways. The 2015;589(22):3379-3387. proposed bifunctionality of OS-9 might assist in the delivery of misfolded glyco- 2. Hebert DN, Molinari M. In and out of proteins to dislocon channels that retrotran- the ER: protein folding, quality control, slocate misfolded glycoproteins from the ER degradation, and related human diseases. to the cytosol. Subsequently, retrotranslo- Physiol Rev. 2007;87(4):1377-1408. cated misfolded glycoproteins in the cytosol 3. Benyair R, Ogen-Shtern N, Lederkre- are successfully degraded upon deglycosy- mer GZ. Glycan regulation of lation. The adjustable catalytic properties of ER-associated degradation through PNGase might be useful for accepting a compartmentalization. Semin Cell Dev wide variety of misfolded glycoproteins. Biol. May 2015;41:99-109. Taken into account the aforementioned examples of bifunctionality during 4. Słomińska-Wojewdózka M, Sandvig K. N-glycosylation biosynthesis, the ability of The role of lectin-carbohydrate interac- glycan-recognizing proteins might be es- tions in regulation of ER-associated sential glycoprotein folding, sensing, sorting, protein degradation. Molecules. and degradation. Moreover, various folding 2015;20(6):9816-9846. diseases i.e. Alzheimer’s diseases, osteopo- 5. Benyair R, Ron E, Lederkremer GZ. rosis, and type 2 diabetes are believed to Protein quality control, retention, and stem from disruption of glycoprotein quality degradation at the endoplasmic reticu- control systems.65 Therefore, insight into lum. Int Rev Cell Mol Biol. Nov glycoprotein bifunctionality may reveal new 2011;292:197-280. opportunities for understanding protein folding diseases. 6. Stanley P. Golgi glycosylation. Cold Spring Harb Perspect Biol. 2011;3(4):a005199. doi: Acknowledgments 10.1101/cshperspect.a005199. We thank to our colleagues and collabora- 7. Parodi A, Cummings RD, Aebi M. Es- tors related Totani group studies. Also these sentials of Glycobiology. Glycans in studies financially supported by JSPS glycoprotein quality control in Chap. 39 KAKENHI No. 23681049, 25560420, 3rd ed. Newyork: Cold spring harbor 16K01938 and 16H06290. laboratory press; 2017 Available from: https://www.ncbi.nlm.nih.gov/books/N BK310274/ [Accessed 4th Aug, 2018].

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