Glycan Remodeling of Human Erythropoietin (EPO) - ACS Publications

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Glycan remodeling of human erythropoietin (EPO) through combined mammalian cell engineering and chemoenzymatic transglycosylation Qiang Yang, Yanming An, Shilei Zhu, Roushu Zhang, Chun Mun Loke, John F. Cipollo, and Lai-Xi Wang ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on May 5, 2017

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Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Qiang Yang1, Yanming An2, Shilei Zhu1, Roushu Zhang1, Chun Mon Loke1, John F. Cipollo2, Lai-Xi Wang1,* 1. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA 2. Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, USA *To whom correspondence should be addressed. E-mail: [email protected]

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ABSTRACT The tremendous structural heterogeneity of N-glycosylation of glycoproteins poses a great challenge for deciphering the biological functions of specific glycoforms and for developing protein-based therapeutics.

We have previously reported a chemoenzymatic glycan remodeling method for

producing homogeneous glycoforms of N-glycoproteins including intact antibodies, which consist of endoglycosidase-catalyzed deglycosylation and novel glycosynthase-catalyzed transglycosylation, but its application to complex glycoproteins carrying multiple N-glycans remains to be examined. We report here site-selective chemoenzymatic glycosylation remodeling of recombinant human erythropoietin (EPO) that contains three N-glycans. We found that the generation of a HEK293S GnT I knockout FUT8 overexpressing cell line enabled the production of an unusual Man5GlcNAc2Fuc glycoform, which could be converted to the core-fucosylated GlcNAc-EPO intermediate acceptor for enzymatic transglycosylation. With this acceptor, homogeneous sialylated glycoform or azide-tagged glycoform were produced using the glycosynthase (EndoF3-D165A) catalyzed transglycosylation. Interestingly, a remarkable site-selectivity was observed in the transglycosylation reactions, leading to the introduction of two N-glycans selectively at the Asn-38 and Asn-83 sites, which was confirmed by a detailed MS/MS analysis of the transglycosylation product. Finally, a different N-glycan was attached at the third (Asn-24) site by pushing the enzymatic transglycosylation with a distinct glycan oxazoline, achieving the site-selective glycosylation modification of the protein. This study represents the first example of site-selective chemoenzymatic glycan engineering of complex glycoproteins carrying multiple N-glycans. KEY WORDS: site-selective transglycosylation, chemoenzymatic glycan remodeling, mammalian cell engineering, erythropoietin (EPO), Endo F3-D165A glycosynthase



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INTRODUCTION Glycosylation can profoundly affect a protein’s intrinsic properties and biological functions, ranging from the folding, stability, and immunogenicity to the cellular functions and in vivo therapeutic efficacy of a given protein 1-9. One major challenge in dealing with glycoproteins is their glycosylation heterogeneity. Natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ only in the structures of pendant glycans, from which pure glycoforms are extremely difficult to isolate by current chromatographic techniques. To address this problem, chemical and biological strategies have been explored for making homogeneous glycoproteins and mimics, including total chemical synthesis with native chemical ligation, chemo-selective ligation, chemoenzymatic synthesis, and glycosylation pathway engineering in host expression systems

10-13

. As part of these

efforts, we have developed a chemoenzymatic glycosylation remodeling method that provides homogeneous glycoforms in two steps: a single step enzymatic deglycosylation using an endoglycosidase and subsequent attachment of a defined N-glycan via a glycosynthase-catalyzed en bloc N-glycan transfer using a glycan oxazoline as the activated donor substrate. Utilizing different substrate selectivity of respective endoglycosidases, we and other labs have developed a tool box of endoglycosidases and related glycosynthases for glycoprotein synthesis and glycosylation remodeling 12-15

. These include the glycosynthases derived from Endo-A (from Arthrobacter protophormiae)

16

,

Endo-M (from fungus Mucor hiemalis) 17,18, and Endo-D (from bacterium Streptococcus pneumoniae) 19

of the glycoside hydrolase (GH) family 85 (GH85), as well as those derived from Endo-S (from

Streptococcus pyogenes)

20

, Endo-S2

21

(from Streptococcus pyogenes), and Endo-F3 (from

Elizabethkingia miricola) 22 of the GH family 18 (GH18). This platform has been especially efficient in synthesis of various glycopeptides and for remodeling of simple glycoproteins such as ribonuclease B. In addition, it has also been successfully applied to the glycan remodeling of therapeutic IgG antibodies due to the discovery of the antibody-specific glycosynthases derived from Endo-S and 3 ACS Paragon Plus Environment

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Endo-S2


20,21,23-26

. Nevertheless, the application of this method for producing homogeneous

glycoproteins with more complex glycosylation patterns remained to be tested. The potential challenges include: the availability of endoglycosidases for efficient deglycosylation of various Nglycans (especially those highly branched N-glycans) from complex glycoproteins; the efficiency of glycosynthase-catalyzed

transglycosylation;

the

limitation

in

substrate

specificity

for

transglycosylation; and the site selectivity of glycosylation remodeling in a multiply glycosylated protein. In this paper, we report a combined approach to chemoenzymatic glycosylation remodeling of human erythropoietin (EPO). EPO is a therapeutic glycoprotein that is widely used for the treatment of anemia by boosting red blood cell production

27

. It is a glycoprotein of 166 amino acid residues that

carries tremendously heterogeneous N-glycans (bi-, tri-, and tetra-antennary with or without sialic acid) at the three conserved N-glycosylation sites (Asn24, Asn38, and Asn83)

28

. It has been demonstrated

that proper glycosylation, particularly sialylation, of EPO is critical for the serum half-life of EPO 29,30. Recently, total chemical synthesis of homogeneously glycosylated EPO carrying 1-3 sialylated Nglycans have been achieved via native chemical ligation of synthetic peptides and glycopeptides

31,32

.

Those endeavors have demonstrated the power of chemical methods in synthesis of homogeneous glycoproteins. Nevertheless, the complexity and multi-step manipulations including the construction of complex glycopeptide building blocks, the often low efficiency in native chemical ligation, and the folding of synthetic glycoproteins still pose great challenge in producing significant amount of pure glycoproteins. We have recently demonstrated that expression of EPO in an engineered HEK293 cell line with overexpression of FUT8 (HEK293S GnT I¯FUT8↑) resulted in the production of an unusual, fully fucosylated Man5GlcNAc2 glycoform of EPO (EPO-M5F, 4) 33. We report in this paper that the fucosylated Man5GlcNAc2 glycoform of EPO could serve as an excellent starting material for endoglycosidase-catalyzed glycan remodeling to provide sialylated or azide-tagged glycoforms of



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EPO. Interestingly, we observed a remarkable site-selectivity of the EndoF3-D165A catalyzed transglycosylation, leading to selective transfer of two sialylated N-glycans at the Asn38 and Asn83 sites. This unusually high site-selectivity allows introduction of a different N-glycan, an azide-tagged N-glycan, at the third (Asn24) glycosylation site, leading to site-selective glycan remodeling of EPO.

RESULTS AND DISCUSSION Inefficient glycan remodeling of complex type and non-fucosylated high-mannose type recombinant EPO by existing endoglycosidases. The goal for this study is to perform glycan remodeling of recombinant EPO to provide homogeneous glycoforms. EPO protein contains three Nglycosylation sites and one O-glycosylation site. The EPO used in present research contains a mutation in S126 O-glycosylation site (Serine to Valine), as previous studies indicated that the O-glycosylation was not essential for either stability or bioactivity of EPO

30

. Our initial attempt to perform directly

glycan remodeling of recombinant EPO expressed from HEK293T cells met with little success. First, we tried to use endoglycosidase to treat complex type EPO to generate a GlcNAc-EPO or Fucα1,6GlcNAc-EPO acceptor. We found that the EPO expressed from HEK293T carries mainly fucoyslated tetra-antennary complex type N-glycans (Figure S1). Treatment of the complex type EPO with Endo-F3 led to no measurable cleavage of N-glycans from the recombinant EPO, as assessed by SDS-PAGE analysis (data not shown). The treatment of the recombinant EPO with Endo-F2 or EndoM could not remove the tetra-antennary N-glycans, either. These results are consistent with their reported substrate selectivity that none of the above endoglycosidase is able to hydrolyze tetra- or higher branched N-glycans. To produce a glycosylated EPO precursor that is cleavable by existing endoglycosidases, we then expressed EPO with high mannose glycan from HEK293T cell in the presence of mannosidase inhibitor kifunensine

34

. As shown in Fig. 1A, MALDI-TOF analysis 5

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indicated major glycans in this glycoform are Man9GlcNAc2 (M9), M8, and M7 (Fig. 1A). This glycoform was processed by Endo-H, resulting in the GlcNAc-EPO acceptor (2) that carries 3 GlcNAc moieties at the 3 N-glycosylation sites. LC-MS analysis confirmed the successful conversion (measured 19969 Da, calculated 19972 Da, as shown in Fig. 1B). Subsequently, we attempted to remodel EPO to complex glycans by using EndoM-N175A mutant, which is known to be able to transfer complex type N-glycan to GlcNAc-protein 16. We selected the sialylated bi-antennary complex type N-glycan oxazoline (S2G2-Oxa) as the donor substrate to transfer it to the GlcNAc-EPO (EPOGn) acceptor (Scheme 1). As shown in Fig 1C, even with the addition of large excess of S2G2-Oxa and high concentration of enzyme (0.3 µg/µl), only one of the three N-glycosylation sites in EPO were partially transferred (found, 21969 Da, calculated, M = 21973 Da). This result indicated that the EndoM-N175 mutant was not efficient at transferring complex type glycan oxazoline to EPO with multiple N-glycosylation sites. The site of transglycosylation in product (3) was not further characterized due to the low efficiency of transglycosylation and the difficulty in isolating the product from the mixtures. In our previous test, Endo-M mutant could not transfer complex type oxazoline to IgG either

35

, although it was able to glycosylate GlcNAc-ribonuclease B

EndoF3-D165A mutant, a glycosynthase derived from Endo-F3

22

16

. We also found that the

, was unable to glycosylate the

GlcNAc-EPO either (data not shown). This is anticipated, as we have previously shown that the EndoF3-D165A mutant was specific for core-fucosylated GlcNAc-protein acceptor, and had only marginal activity on non-fucosylated GlcNAc acceptor 22.



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Scheme 1. Glycan modeling of GlcNAc-EPO (EPO-Gn) acceptor with EndoM-N175A glycosynthase. The positions of three N-glycosylation sites were labeled in EPO-HM (1)

Fig.1. Inefficient glycan remodeling of EPO-Gn acceptor with EndoM-N175A glycosynthase. A) MALDI-TOF analysis of PNGaseF-released glycan from EPO-HM. B) LC-MS analysis of EPO-Gn. C) LC-MS analysis of result of transglycosylaton reaction.

Generation of core-fucosylated EPO glycoform suitable for EndoF3 glycosynthasecatalyzed transglycosylation. Having confirmed that the non-fucosylated acceptor GlcNAc-EPO (2) could not be effectively transferred with an existing glycosynthase, we next sought to generate corefucosylated EPO glycoform, Fucα1,6GlcNAc-EPO (EPO-GnF, 5), which might serve as a good 7 ACS Paragon Plus Environment

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acceptor substrate for EndoF3 glycosynthase-catalyzed transglycosylation. Recently, we observed unexpected high corefucosylation of Man5GlcNAc2 (M5) glycan (more than 50%) of EPO produced from a HEK293S GnTI knockout cell line

33

. To characterize the underlying mechanism and to

increase the fucosylation level of EPO-M5 as a potential starting material for glycan remodeling, we have generated a 293S GnTI¯FUT8↑ cell line by lentiviral-mediated gene transfer, and found that expression of EPO from this cell line yielded almost 100% homogenous M5F glycan

33

. This

successful generation of this unusual glycoform prompted us to explore it to obtain the Fucα1,6GlcNAc-EPO as the key precursor for EPO glycan remodeling. For the purpose, we first tested recombinant Endo-H and Endo-A for deglycosylation of this EPO intermediate (Scheme 2). As shown in Fig. 2A, Endo-H could effectively cleave three M5F on EPO, generating EPO-GnF (5). LC-MS analysis confirmed the complete deglycosylation (deconvoluted, 20406 Da, calculated, M = 20410 Da, Fig. 2B; m/z profile, Fig. S2A). We found that Endo-A could also efficiently process EPO-M5F (4) to give the homogeneous EPO-GnF intermediate (5) (data not shown).

Fig.2. Generation of the EPO-GnF acceptor. A) SDS-PAGE analysis. Lane 1, EPO-M5F; lane 2, EPOGnF. B) LC-MS analysis of EPO-GnF.

Glycan remodeling of EPO with homogenous sialylated complex type glycan and azidetagged glycan. Having successfully acquired the EPO-GnF (5) acceptor, we tested the transfer of



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S2G2-oxa by the EndoF3-D165A glycosynthase (Scheme 2). Incubation of EPO-GnF (5) with 60 molar equivalent of the glycan oxazoline (20 equivalents for each glycosylation site) and 0.05 µg/µl concentration of glycosynthase for 30 min gave a single product (6) that appeared as a band about 4 KDa (equivalent to size of two S2G2 glycans) larger than that of the starting material on SDS-PAGE (Fig. 3A). Meanwhile, the band of starting material EPO-GnF totally disappeared, indicating full conversion. LC-MS analysis showed that the transferred product (6) had a deconvoluted molecular weight of 24411 Da (Fig. 3B; m/z profile in Fig. S2B, supporting information), which corresponds to an addition of two S2G2 glycans (calculated MW 24413). The transfer did not proceed in such a condition for another 30 min (Fig. 3A, lane 3). In this case, a high regio-selectivity was achieved, and the glycan-remodeled EPO glycoform (6), which carries two sialylated N-glycans selectively at the N38 and N83 sites (as determined by tandem MS analysis below) and a His tag at the C-terminus, was readily isolated by Ni-NTA affinity chromatography and its purity was confirmed by ESI-MS analysis (Fig. 3B). When more oxazoline (totally 180 ea) and enzyme (final concentration 0.2 µg/µl) were added and

incubation time was extended, a second band that ca. 6 KDa larger than that of the starting material (EPO-GnF) appeared on SDS-PAGE (Fig. 3C, lane 4). LC-MS confirmed that the second band was the transglycosylation product (7) that carries three sialylated S2G2 N-glycans (found, 26414 Da, calculated M = 26416 Da, Fig. 3D). The transglycosylation result illustrated that, among the three glycosylation sites of EPO, two sites appear much more accessible to the glycosynthase than the third site. With extended reaction (“push” condition), the transfer of S2G2-oxa could reach approximately 60% on the third site (100% on other two other sites). The high efficiency of transfer is in sharp contrast to EndoM-N175A catalyzed transglycosylation with GlcNAc-EPO as the acceptor.


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Fig.3. Efficient remodeling of EPO to homogenous complex glycoform or azide-tag glycoform with EPO-GnF as starting material. A) SDS-PAGE analysis of EndoF3-D165A catalyzed transglycosylation of sialylated complex type glycan. Lane 1, EPO-GnF; Lane 2, EPO-GnF transferred with S2G2-Oxa in 30 min; Lane 3, transglycosylation at 1 hr; Lane 4, transglycosylation overnight. B) LC-MS analysis of EPO-(S2G2)2. C) LC-MS analysis of EPO-(S2G2)2-3. D) LC-MS analysis of EPO-(M3N3)2. The major peak of 21885 is EPO transferred with two M3N3 glycans. The minor peak of 21145 is the product of transfer with one M3N3 glycan, while 22625 Da is transferred with three M3N3 glycans

In addition to sialylated glycan, we also tested if EndoF3-D165A could transfer azide-tagged Man3GlcNAc2 (M3N3) glycan to EPO-GnF acceptor (5) (Scheme 2). As shown in Fig. 3D, a major peak of 21885 Da (8), representing transfer of two M3N3 glycans (calculated 21890 Da), appeared after the transfer reaction in LC-MS analysis. This site-selective transfer is very similar to the transfer reaction of sialylated glycan. The EPO-(M3N3)2 glycoform could be further modified to add polysialylated oligosaccharides to extend the serum half-life of EPO by click chemistry. Functional group could also be introduced to help study the activity of EPO.



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Scheme 2. Glycan remodeling of Fucα1,6GlcNAc-EPO (EPO-GnF) acceptor with EndoF3-D165A glycosynthase

Determination of the transglycosylation site selectivity in the product (6) by LC-MS analysis. The interesting site selectivity in the EndoF3-D165A glycosynthase catalyzed transglycosylation of the EPO-GnF (5) that carries three Fucα1,6GlcNAc moieties prompted us to perform a detailed MS/MS of the transglycosylation product (6) to define the transferred sites. After digestion of 6 with endoproteinase Glu-C and trypsin, the glycopeptides generated were enriched via hydrophilic interaction on Amide 80 HILIC resin and subjected to MS/MS analysis. Representative spectra from glycopeptides containing glycosylation site N38 (A) and N83 (B) obtained from digestion of the glycan-remodeled EPO (6) were shown in Figure 4.


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Fig.4. LC-MSE analysis of the glycopeptides derived from EPO to deduce the glycosylation site occupancy. A) analysis of glycopeptide containing N38 glycosylation site. B) analysis of glycopeptide containing N83 glycosylation site. The data is the representative of three independent experiments. The green peak in each spectrum was the intact glycopeptide ion signal. The peptide sequence was determined by a series of b- and y- ions, which are indicated by blue and red lines on the left part of the spectra. The oxonium ions at m/z 204.09, 366.14, 209.10 and 657.24 indicate the presence of HexNAc, Hex-HexNAc, NeuAc and HexNAc-Hex-NeuAc, respectively. They were used as diagnostic markers to assess whether the predicted glycoform composition was correct. The fragment ions on the right half of the spectra were the glycosidic bond cleavage products. They were used to derive the sequence and composition of the glycan moiety as shown in Figure 4. For the glycopeptide with N38, the ion at m/z 1090.56 was the peptide NITVPDTK plus the aglycone most GlcNAc residue. It was used to verify peptide identity. The ion at m/z 1236.62 was the peptide bearing the GlcNAc+Fuc 12 ACS Paragon Plus Environment


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structure, indicating the presence of a core fucose moiety. We also annotated the inferred glycan sequences for some fragment ions in the figure. For the glycopeptide with N83, the peptide plus the GlcNAc residue was at m/z 2562.32 and a series of glycosidic fragments were annotated in the spectrum. No glycopeptide containing N24 site was detected, which suggested that this site was not transglycosylated thus was not trapped by the Amide 80 HILIC resin. Taken together, the LC-MSE analysis demonstrated that among the three sites of EPO, N38 and N83 were the preferred sites for transglycosylation by Endo-F3 glycosynthase, while the N24 site was much less reactive. Site-selective glycan remodeling of EPO. The discovery of the selectivity in transglycosylation reactivity of the three glycosylation sites of EPO raised an exciting possibility to perform site-selective transglycosylation of this protein, transferring one glycan to two sites (N38 and N83), and adding a different glycan to the third site (N24). We designed the reaction strategy as following: 1) transferring S2G2-oxa to site N38 and N83 by EndoF3-N165A in a controlled reaction condition, resulting in glycoform 6, 2) removing EndoF3-N165A and unreacted S2G2-oxa from reaction mixture, 3) defucosylation of glycoform 6, 4) adding the second Man3GlcNAc-azide oxazoline by Endo-A glycosynthase (Scheme 3). Successful implementation of this strategy led to the synthesis of glycoform 9 that carries two sialylated N-glycans at N38 and N83 sites and an azide tagged Man3GlcNAc2 glycan at the N24 site (Fig. 5). Briefly, glycoform 6 was prepared by controlled transglycosylation of 5, giving the glycoform (6) that carries two sialylated N-glycans at N38 and N83 sites. To introduce an azide-modified Man3GlcNAc glycan using Endo-A as the enzyme

36

, which

could not use core-fucosylated GlcNAc-protein as the acceptor substrate, the fucose residue in the Fucα1,6GlcNAc at the N24 site of 6 was removed by the treatment with the α1,6 fucosidase from Lactobacillus casei. It was found that this α-fucosidase was able to remove the fucose residue in the disaccharide Fucα1,6GlcNAc moiety (at N24), but not on the core fucose in the context of full length 13 ACS Paragon Plus Environment

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N-glycans (at the N38 and N83 sites), probably due to steric hindrance. Similar phenomenon with this fucosidase was observed in the glycosylated and deglycosylated Fc domain of IgG antibodies including rituximab (unpublished data). A recent report also showed that the α-fucosidase from Bacteroides fragilis had similar selectivity in defucosylation

37 21

.

Finally, Endo-A catalyzed

transglycosylation of glycoform 9 with the azide-Man3GlcNAc oxazoline gave the novel glycoform 10. In the present case, we used an immobilized EndoF3-D165A for the first step transglycosylation in order to facilitate the removal of EndoF3-D126A from the reaction system. The glycosynthase was immobilized to activated agarose bead containing aldehyde group through reductive amination. Scheme 3. Site-selective glycan remodeling of EPO-GnF

As shown in Fig 5A, the use of the immobilized glycosynthase led to a slight reduced yield of transglycosylation, giving EPO-(S2G2)2 (6) as the major product, together with some unreacted EPOGnF (5), partially transferred EPO-S2G2, and fully transferred EPO-(S2G2)3 (7) as the minor byproducts. Selective defucosylation at the N24 site with the a-fucosidase gave glycoform 9 as the major product (major peak boxed in red in Fig.5B; found, 24265 Da, calculated, M = 24266 Da). The minor peaks are EPO-S2G2 with fucoses on two GnF moiety removed (22116 Da) and intact glycoform 7 (26414 Da). The final transfer was performed by addition of EndoA and azide-Man3GlcNAc (M3N3) 14 ACS Paragon Plus Environment


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oxazoline. As illustrated in Fig 5C, a new major peak (boxed in red) was shown, which was the expected final product EPO-(S2G2)2-M3N3 (glycoform 10, found, 25005 Da, calculated, M = 25005 Da). The glycan transfer reached approximately 50% after two-hour reaction. A minor peak of 23595 Da was the defucosylated EPO-S2G2 transferred with two M3N3. The results suggest that it is possible to perform site-selective glycan remodeling among the three N-glycans in EPO. In this case, the last enzymatic transglycosylation was not optimized and the reaction was incomplete. The EPO was readily separated from the glycans and enzyme via the Ni-NTA affinity chromatography. However, it was still a mixture of the precursor 6 and the glycan remodeled final product, EPO-(S2G2)2-M3N3 (10). While we did not attempt to purify the final product from the mixtures, the purification could be achieved through lectin-affinity chromatography, such as the wheat germ agglutinin (WGA) that can selectively bind the Man3GlcNAc2 moiety. In practice, it would be ideal to push the reaction to completion so that a tedious separation of glycoforms mixtures would be avoided. The introduction of an azide-modified N-glycan at a selective site will allow

further selective modifications such as introduction of a fluorescent probe or another tag for functional studies.

Fig.5. Site-selective glycan remodeling of EPO. A) LC-MS analysis of EPO-(S2G2)2. B) LC-MS analysis of defucosylated EPO-(S2G2)2. C) LC-MS analysis of EPO-(S2G2)2-M3N3.


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The site-selectivity of EPO transglycosylation might be due to the difference in steric hindrance at different glycosylation sites. N38 and N83 sites of EPO seem more exposed than N24 sites, permitting selective introduction of two N-glycans at these two sites, and a different N-glycan at the N24 site. Previous site-specific glycosylation profiling of recombinant EPO indicated that the glycans attached at the N38 and N83 sites were mainly tetra-antennary glycans while that at the N24 site was a mixture of bi-, tri-, and tetraantennary glycans

38

. The data also imply that the N38 and N83 sites might be more accessible for enzyme

processing to give the fully processed and highly branched tetra-antennary N-glycans as the major glycoforms, while the N24 site might be less accessible for enzymatic processing to yield the less branched glycoforms. These results appear to be consistent with our observation of site selectivity in the enzymatic glycan remodeling of EPO. As to the glycan remodeled EPO described in this work, the two sialylated N-glycans

introduced at N38 and N83 could help to sustain the solubility and stability of the glycoprotein

32

,

while the azide modified N-glycan attached at the N24 site could be used to introduce other functional groups via click chemistry, such as a fluorescent tag or other chemical probe for functional studies of EPO. While the generality of this approach remains to be tested with additional multiply glycosylated proteins, the present discovery suggests that a combination of the distinct accessibility of glycosylation sites, the difference in substrate specificity of endoglycosidases, and the nature of the N-glycans could achieve site-selective modification of N-glycans in the context of a complex glycoprotein.

CONCLUSION A chemoenzymatic glycan remodeling of erythropoietin (EPO) that carries 3 N-glycans is described. The combination of mammalian cell engineering and in vitro chemoenzymatic glycan remodeling represents a novel platform for producing homogeneous glycoforms of erythropoietin. An important finding from the present study is the discovery of highly site-selective transglycosylation of the three 16 ACS Paragon Plus Environment


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N-glycosylation sites in EPO, enabling selective introduction of different N-glycans in the glycoprotein. The present study represents the first example of site-selective glycan modification of a therapeutic protein carrying multiple N-glycans, which remains a challenging task and deserves further explorations. MATERIALS AND METHODS Cell culture. Human HEK293T cells were cultured in suspension in FreeStyleTM F17 serum-free medium supplemented with GlutaMAX (Life Technologies), incubating at 37°C, 8% CO2, and shaking of 140 rpm/min. The cells were passaged every 3-4 days with the initial seeding density of 3 ×105 cells per ml. The culture of HEK293S GnTI¯FUT8↑followed the procedures previously reported by Yang et al. 33. Expression and purification of EPO-HM and EPO-M5F (4) from human cell ine. The high mannose type EPO (EPO-HM) was produced in the HEK293T cells by transient transfection in the presence of 5 µg/ml of kifunensine (Cayman Chemical, Ann Arbor, Michigan), a mannosidase inhibitor. The EPOM5F (4) was produced in HEK293S GnTI¯FUT8↑ cells. The construction of EPO-S126V expressing plasmid, the transfection, and purification procedures were as described by Yang et al. 33. Expression and purification of endoglycosidase and glycosynthase from E. coli. The expression and purification of Endo-A and its mutants followed protocol as described by Huang et al.16. EndoMN175A mutant was prepared according to the reported protocol

17

. EndoF3-D165A mutant was made

following the previously reported procedures 22. AlfC, α1,6-fucosidase from Lactobacillus casei was prepared as described in a previous report 39. Generation of EPO-Gn or EPO-GnF acceptor. EPO-Gn acceptor was created by treatment of EPOHM with Endo-Hf (New England Biolabs). 200 µg of EPO-HM was treated with 1000 u of Endo-Hf in 17 ACS Paragon Plus Environment

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100 µl of PBS buffer, pH 7.4. The mixture was incubated at 37°C for one hour. EPO-GnF acceptor was generated in a similar manor. 400 µg of EPO-GnF was processed by 2000 u of Endo-Hf in 200 µl of PBS buffer. Synthesis of oxazolines. Biantennary sialylated complex glycan oxazoline (S2G2-Oxa) was synthesized according to the previously reported protocol

40

. Azido tagged Man3GlNAc2 oxazoline (M3N3-Oxa)

was prepared following the published procedures 36. Glycan remodeling of EPO to homogenous complex glycoform. In the initial trial, the GlcNAc-EPO was transferred with biantennary sialylated complex glycan oxazoline (S2G2-Oxa) by EndoM-N175A glycosynthase. 100 µg of GlcNAc-EPO is mixed with 15 µg of EndoM-N175A, 2 mg of S2G2-Oxa, in 50 µl of PBS buffer, pH 7.4. The reaction mixture was incubated at 30°C overnight. Aliquot was taken at different time points to monitor the reaction with either SDS-PAGE or LC-MS. The EndoM mutant catalyzed transfer turned out to be quite inefficient. Next, we tried transfer S2G2-Oxa to EPO-GnF acceptor with EndoF3-D165A glycolsynthase. The transfer was carried out in two different conditions: controlled or “push” reaction. In controlled reaction condition, 200 µg of EPO-GnF was mixed with 1.18 mg S2G2-Oxa (60 ea), 5 µg of EndoF3-D165A glycosynthase (final concentration 0.05 µg/µl), in 100 µl of PBS buffer, pH 7.4. The mixture was incubated at 30°C for 15-30 min. Under such condition, the transglycosylation product is EPO-(S2G2)2, EPO transferred with two S2G2 glycan. In the “push” condition, 50 µg of EPO-GnF was mixed with 300 µg (60 ea) of S2G2-Oxa, 2.5 µg of EndoF3-D165A (final concentration 0.1 µg/µl), in 25 µl of PBS buffer, pH 7.4. After 30 min of incubation at 37°C, another aliquot of S2G2-Oxa (120 eq) and 2.5 ug of EndoF3-D165A (final concentration 0.2 µg/µl) was added and reaction was extended for two hours to overnight. In this condition, the main transglycosylation product is EPO with three S2G2 glycan.



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Transfer of azide-tagged glycan to Fucα1,6GlcNAc-EPO (EPO-GnF) acceptor by EndoF3-D165A glycosynthase. 50 µg of EPO-GnF was mixed with 114 µg azide-tagged Man3ClcNAc oxazoline (M3N3-Oxa) (60 ea), 2.5 µg of EndoF3-D165A glycolsynthase (final concentration 0.1 µg/µl), in 25 µl of PBS buffer, pH 7.4. The mixture was incubated at 30°C for 30 min. Afterwards, another aliquot of M3N3-Oxa (60 ea) and 2.5 ug of EndoF3-D165A (final concentration 0.2 µg/µl) was added and reaction was extended for additional 30 min. Site-selective glycan remodeling of EPO. In first step, the EPO-GnF acceptor was transferred with S2G2 glycan to N38 an N83 glycosylation sites. To facilitate its removal after reaction, the EndoF3D165 glycosynthase was immobilized to AminoLink Agarose bead (ThermoFisher Scientific) to a final concentration of 1mg/ml resin according to manufacturer’s protocol. 15 µg of EPO-GnF was mixed with 176 µg S2G2-Oxa (120 ea), 30 µl of EndoF3-D165A-agarose, in a 47 µl suspension mixture in 50 mM sodium acetate buffer, pH 6.0. The suspension mixture was incubated at 37°C for 60 min in a head-to-end rotator. After LC-MS confirmation of formation of major EPO-(S2G2)2 product, the immobilized EndoF3 mutant was removed by gentle centrifugation (100 g × 2 min). An overnight dialysis with a Slide-A-Lyzer™ MINI Dialysis Devices (ThermoFisher) at 4°C was performed to remove unreacted oxazoline and change the buffer to PBS, pH 7.4. In second enzymatic reaction, defucosylation was performed by adding 1 µg of AlfC (α1,6-fucosidase) to 15 µl of EPO-(S2G2)2 intermediate product and incubating at 37°C for 12 hr. The completion of defucosylation was confirmed by LC-MS. In the final step, the transfer of M3N3-Oxa to the N24 glycosylation site was catalyzed by EndoA. The defucosylated EPO-(S2G2)2 was mixed with 3 µg of EndoA (final concentration 0.2 µg/µl), 100 µg of M3N3-Oxa (180 ea), then incubated at 37°C for 90 min. The reaction was pushed with adding another 180 ea of M3N3-Oxa and incubating for 90 more min. The final transglycosylation product was analyzed with LC-MS. 19 ACS Paragon Plus Environment

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Mass spectrometry analysis of intact EPO and released N-glycan. Intact EPO was analyzed with Liquid Chromatography Electrospray Mass spectrometry (LC-ESI-MS) on an ExactivePlus OrbiTrap system (Thermo Scientific) with a C8 column (Poroshell 300SB-C8, 1.0×75mm, 5 µm, Agilent) with 5-90% acetonitrile linear gradient in 6 min. The raw data was deconvoluted with MagTran (Amgen). The PNGaseF-released N-glycan was analyzed with an Autoflex III Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics). The release, clean-up, and analysis of glycan followed the previously reported procedures 33. LC-MSE analysis of the glycopeptides from EPO-(S2G2)2. To identify two transferred glycosylation sites, EPO-(S2G2)2 protein was digested with endoproteinase Glu-C and trypsin. Intact glycopeptides were enriched via solid phase extraction with TSKgel Amide 80 HILIC resin according to our previous report 41. The glycopeptides were injected onto a C18 column (HSS T3 nanoACQUITY column 75µm i.d. x 100 mm, 1.8 µm particle, Waters Corporation) and the effluent was introduced into a Waters SYNAPT™ G2 HDMS system (Waters Corp. Milford, MA). The instrument was set to perform MSE experiments. The low energy precursor ion scan was performed at 4 eV. The elevated collision energy fragmentation scam was performed in a linear ramp from 15 to 45 eV. NanoLC/MSE is a relatively new MS technique, which collects MS data in an alternating scan mode between low and elevated collision energies. The precursor ion data are collected during the low energy MS scan, and then the collision energy is ramped linearly over a range of voltages in the dissociation stage to fragment the precursor ions. The data produced during the experiment are used to reconstruct MS/MS spectra without the bias associated with data-dependent ion scanning. The process can be optimized not only to fragment the glycan portion but also to generate abundant b- and y-ions from the peptide backbone. Therefore, the MSE spectra are rich in fragment ions from both glycoside and peptide moieties of the glycopeptides analyzed 42. 20 ACS Paragon Plus Environment


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CONFLICT OF INTEREST The authors declare no conflicts of interest with the contents of this article. ACKNOWLEDGEMENT We thank members of the Wang lab for helpful discussions and technical assistance. This work was supported by the National Institutes of Health (NIH grant R01 GM080374). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. Supplementary figures for ESI-MS profiles of recombinant and glycan-remodeled EPO.


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