Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Article abstract of DOI:10.1021/jacs.7b07906

Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multistep chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide but also to high-mannose and complex-type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

Full text of DOI:10.1021/jacs.7b07906

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Article
Designer #1,6-Fucosidase Mutants Enable Direct Core
Fucosylation of Intact N-Glycopeptides and N-Glycoproteins
Chao Li, Shilei Zhu, Christopher Ma, and Lai-Xi Wang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07906 • Publication Date (Web): 09 Oct 2017
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Designer α1,6ꢀFucosidase Mutants Enable Direct Core Fucosylation
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of Intact NꢀGlycopeptides and NꢀGlycoproteins
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Chao Li, Shilei Zhu, Christopher Ma, Lai-Xi Wang*
Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive,
College Park, Maryland 20742, USA
*Corresponding Author: email: wang518@umd.edu
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ABSTRACT
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Core fucosylation of Nꢀglycoproteins plays a crucial role in modulating the biological functions
of glycoproteins. Yet, the synthesis of structurally wellꢀdefined, coreꢀfucosylated glycoproteins
remains a challenging task due to the complexity in multiꢀstep chemical synthesis or the inability
of the biosynthetic α1,6ꢀfucosyltransferase (FUT8) to directly fucosylate fullꢀsize mature
Nꢀglycans in a chemoenzymatic approach. We report in this paper the design and generation of
potential α1,6ꢀfucosynthase and fucoligase for direct coreꢀfucosylation of intact Nꢀglycoproteins.
We found that mutation at the nucleophilic residue (D200) did not provide a typical
glycosynthase from this bacterial enzyme, but several mutants with mutation at the general
acid/base residue E274 of the Lactobacillus casei α1,6ꢀfucosidase, including E274A, E274S, and
E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex
Nꢀglycopeptides and intact glycoproteins by using αꢀfucosyl fluoride as a simple donor substrate.
Studies on the substrate specificity revealed that the α1,6ꢀfucosidase mutants could introduce an
α1,6ꢀfucose moiety specifically at the Asnꢀlinked GlcNAc moiety not only to GlcNAcꢀpeptide,
but also to highꢀmannose and complex type Nꢀglycans in the context of Nꢀglycopeptides,
Nꢀglycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of
homogeneous, coreꢀfucosylated Nꢀglycopeptides and Nꢀglycoproteins that are hitherto difficult
to obtain for structural and functional studies.
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INTRODUCTION
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Asparagineꢀlinked glycosylation, namely the Nꢀglycosylation, is one of the most prevalent
posttranslational modifications of proteins in mammals, which plays important roles in
modulating the intrinsic properties and biological functions of the underlying proteins 1,2. For
example, the Nꢀglycans attached can have a profound effect on protein’s folding, stability,
antigenicity, and immunogenicity 2,3. On the other hand, the Nꢀglycans can directly participate in
a variety of biological recognition processes, including cell adhesion, hostꢀpathogen interaction,
cancer metastasis, and immune response 1,4ꢀ9. While all mammalian Nꢀglycans share a common
oligosaccharide core structure, further decoration of the core, such as sialylation and fucosylation,
adds another level of structural diversity in modulating biological functions.
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Coreꢀfucosylation, the attachment of an α1,6ꢀlinked fucose to the innermost,
asparagineꢀlinked Nꢀacetylglucosamine (GlcNAc) moiety in the Nꢀglycans, is an important
modification of Nꢀglycoproteins. Compelling data have shown that coreꢀfucosylation of
glycoproteins regulates a wide range of cellular functions. For example, many studies have
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revealed that increased core fucosylation is often associated with cancer progression
.
AFPꢀL3, the coreꢀfucosylated αꢀfetoprotein has been approved by regulatory agency as a
biomarker for hepatocellular carcinoma, a major form of liver cancer 13. The roles of
coreꢀfucosylation in development has been demonstrated by the experiments that knockout of
FUT8 gene in mouse models induces severe growth retardation and death during postnatal
development 14. Core fucosylation also directly modulates the biological activities of
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glycoproteins, such as the antibody dependent cellular cytotoxicity (ADCC) of therapeutic
monoclonal antibodies 15, the signaling functions of growth factor receptors and adhesion
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molecules 14,16ꢀ20, and the antigen recognition of IgG B cell receptors . On the other hand,
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structural studies have suggested that coreꢀfucosylation could affect the conformations of
Nꢀglycans 22,23
.
Given the difficulties to isolate homogeneous glycoforms from natural sources, synthesis of
structurally wellꢀdefined, coreꢀfucosylated glycopeptides and glycoproteins is essential for
various glycomics studies aiming to further decipher the structural and functional impact of
coreꢀfucosylation 24ꢀ26. In animals and humans, coreꢀfucosylation is catalyzed solely by the
mammalian α1,6ꢀfucosyltransferase, FUT8 27,28. However, FUT8 has a very strict substrate
specificity, requires the presence of a free GlcNAc at the α1,3ꢀlinked mannose arm in the
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Nꢀglycan as the substrate and usually is unable to fucosylate fullꢀsize mature Nꢀglycans
.
Only until recently we have provided the first examples showing that FUT8 could catalyze in
vitro fucosylation of some highꢀmannose Nꢀglycans lacking a free GlcNAc at the α1,3ꢀlinked
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mannose arm when the glycan is present in an appropriate protein or other context . This strict
substrate specificity makes the α1,6ꢀfucosyltransferase of limited usefulness for chemoenzymatic
synthesis. On the other hand, chemical synthesis of coreꢀfucosylated Nꢀglycopeptides and
Nꢀglycoproteins is more complex than the synthesis of those nonꢀfucosylated glycoconjugates,
due to the difficulty in control of the αꢀstereoꢀselectivity in glycosylation and the acidꢀlabile
nature of the α1,6ꢀfucosidic linkage 34,35. A method for direct fucosylation of intact
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glycopeptides and glycoproteins is highly desirable. We report in this paper the discovery of
novel mutants derived from Lactobacillus casei αꢀfucosidase, which are able to use αꢀfucosyl
fluoride as the substrate for direct coreꢀfucosylation of intact Nꢀglycopeptides and
Nꢀglycoproteins without product hydrolysis. We found that the rationally designed mutants,
E274A/S/G derived from Lactobacillus casei αꢀfucosidase carrying a single mutation at the
general acid/base residue (E274) acted as an efficient fucoligase and were able to fucosylate a
wide variety of substrates including large synthetic Nꢀglycopeptides, natural Nꢀglycoproteins,
and intact monoclonal antibodies, paving a way to obtain coreꢀfucosylated glycopeptides and
glycoproteins for structural and functional studies.
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RESULTS AND DISCUSSION
Design. The goal of this study is to generate glycosidase mutants capable of using simple
glycosyl donors for direct coreꢀfucosylation of intact Nꢀglycopeptides and Nꢀglycoproteins,
which could not be achieved by the catalysis of the biosynthetic pathway α1,6ꢀfucosyltransferase
(FUT8) because of its strict substrate specificity. There are two general glycosidase engineering
strategies that may convert a glycosidase into a synthetically useful mutant. One is the
glycosynthase concept through siteꢀdirected mutagenesis at the critical nucleophilic residue of a
retaining glycosidase to generate a mutant that is devoid of hydrolysis activity but can take an
activated glycosyl donor (usually a glycosyl fluoride) with an opposite anomeric configuration
for transglycosylation 36ꢀ38. Glycosynthases derived from several GH family glycosidases have
been successfully created using this strategy 39ꢀ43. The other is the glycoligase approach, first
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developed by Withers and coꢀworkers, in which the general acid/base residue of a retaining
glycosidase is mutated to eliminate the hydrolysis activity, and the enzymatic transglycosylation
is enabled by using an activated glycosyl donor with the same anomeric configuration 44ꢀ47. For
βꢀglycosynthases derived from the corresponding retaining βꢀglycosidases, the readily
synthesized and relatively stable αꢀglycosyl fluorides have become the common glycosyl donor
substrates 39,40. However, the evaluation of the transglycosylation activity of potential
αꢀfucosynthases usually requires a βꢀfucosyl fluoride, which is quite unstable in aqueous
solution and will be hydrolyzed spontaneously with a halfꢀlife of ca. 20 min 48. Previously
several α1,2ꢀ and α1,3ꢀ1,4ꢀfucosynthases have been generated from the Bifidobacterium bifidum
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αꢀLꢀfucosidases and examined for enzymatic transfucosylation of glycoconjuagtes, but the
dependence on the use of the highly unstable βꢀglycosyl fluoride renders these mutants less
attractive for synthetic purpose 48ꢀ51. As an alternative approach, Moracci and coꢀworkers have
shown that the stable βꢀfucosyl azide could serve as a glycosyl donor for the αꢀfucosynthases
derived from the hyperthermophilic archaeon Sulfolobus solfataricus αꢀ
βꢀgalactosyl azide was also successfully applied as a substrate for an αꢀgalactosynthase 53,54
L
ꢀfucosidase 52. The
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Despite these successes, no α1,6ꢀfucosynthase or any αꢀfucoligase has been reported so far.
To explore this possibility, we started our pursuit by choosing the α1,6ꢀfucosidase from
Lactobacillus casei (AlfC) as the model enzyme, which was shown to hydrolyze specifically
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α1,6ꢀfucosidic linkage and was recently reported to have transglycosylation activity, capable
of making Fucα1,6GlcNAc disaccharide, using pꢀnitrophenyl αꢀfucopyranoside as the donor
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substrate and GlcNAc as the acceptor 56,57. Nevertheless, the wild type enzyme also hydrolyzes
the disaccharide product rapidly leading to low synthetic efficiency. Moreover, it has been
unclear if this enzyme could act on more complex substrates for transglycosylation other than a
simple GlcNAc substrate. Thus, our design is to generate potential glycosynthase and glycoligase
mutants from the Lactobacillus casei α1,6ꢀfucosidase, and to test their ability to coreꢀfucosylate
various acceptor substrates (Figure 1). For evaluating the potential glycosynthase mutants, we
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would use the stable βꢀfucosyl azide (1) as the substrate. In addition, since βꢀfucosyl fluoride
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is highly unstable with a halfꢀlife of ca. 20 min in a neutral aqueous solution , we sought to
synthesize the 2ꢀdeoxyꢀ2ꢀfluoroꢀβꢀfucosyl fluoride (2) as a more stable substrate to test the
potential αꢀfucosynthase mutants. For evaluating the potential glycoligase mutants, the αꢀfucosyl
fluoride (3) would be used as a donor substrate.
Alignment of Amino acid sequence with GH29 α-fucosidases. GH29 retaining
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αꢀLꢀfucosidases catalyze the removal of nonꢀreducing terminal Lꢀfucose residues in the α1,2,
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α1,3, α1,4, or α1,6ꢀglycosidic linkages in oligosaccharides and glycoconjugates. In order to
determine the key catalytic residues of AlfC αꢀfucosidase, several αꢀfucosidases belonging to the
GH29 family were chosen in alignment of amino acid sequences, due to their distinct substrate
specificities and positions of the two catalytic residues clarified in previous reports 58ꢀ61. The
results were shown in Figure 2. All the positions of catalytic nucleophile, an aspartate residue,
turned out to be completely aligned up among the five αꢀfucosidases including AlfC (Indicated
by blue arrowhead and box in Figure 2). It has been previously shown that the nucleophile
residue is fully conserved across the GH29 family 62. Thus residue D200 in AlfC which is
aligned with the nucleophilic residues of the other GH29 family enzymes was very likely the
nucleophile residue of the AlfC enzyme. The positions of general acid/base seemed more
complicated, as these residues scattered around in these enzyme sequences (Indicated by purple
box in Figure 2). The glutamate residue E274 of AlfC aligned with the E289 of Homo sapiens
A1 (Hfuc), while no polar amino acid residue of AlfC showed up at the positions of the general
acid/base residues in other αꢀfucosidases. Interestingly, both Hfuc and AlfC turned out to be
α1,6ꢀfucosidases. Considering the alignment results and the substrate specificity, we assumed
that the E274 residue could be the catalytic general acid/base residue for the AlfC enzyme.
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Generation of potential αꢀfucosidaseꢀbased glycosynthases and glycoligases. Following
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the glycosynthase concept proposed by Withers and coꢀworkers , we performed siteꢀdirected
mutagenesis at the identified nucleophile in the AlfC α1,6ꢀfucosidase, D200, to generate selected
mutants, including D200G, D200S, D200A, and D200T. Similarly, selected mutants at the
putative general acid/base residue, E274, were generated to provide potential glycoligases,
including E274A, E274S, E274G, and E274D (Table S1). These mutants were expressed as a
fusion protein with a Cꢀterminal Vibrio cholera MARTX toxin cysteine protease domain (CPD)
carrying a 10× His tag, as previous experiments have demonstrated that the CPD tag can enhance
the solubility and stability of recombinant proteins 63ꢀ66. All these mutants were efficiently
expressed in E. coli with a relatively high yield (more than 40 mg/L) and were readily purified
by NickelꢀNTA affinity chromatography 63. We then tested the hydrolysis activity of these
mutants together with wild type AlfC using pꢀnitro αꢀfucoside (pNPFuc) as the substrate. As
expected, all these mutants, except E274D, showed only trace residual hydrolysis activity due to
the mutations at the critical residues, while (Figure S1). This study confirms that the D200
residue is the nucleophile and that the E274 residue is most likely the general acid/base residue.
In the case of E294D, the similarity between the Glu and Asp suggests that the Asp residue in
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place of the E274 could still play the role of the Glu residue as the general acid/base for
promoting hydrolysis of the substrate.
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Assessment of the mutants as potential glycosynthases or glycoligases. First, we
examined the D200 mutants as potential glycosynthases using two types of activated fucosyl
donors with opposite anomeric configuration as the donor substrates. As βꢀglycosyl fluorides are
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quite unstable and are subjected to spontaneous hydrolysis in aq. solutions , we synthesized
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2ꢀdeoxyꢀ2ꢀfluoroꢀβꢀfucosyl fluoride (2) as a more stable substrate (Scheme S1) . We tested its
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stability in an aqueous buffer and found that the halfꢀlife of 2 was about 6 h at 37 C in a
phosphate buffer (100 mM, pH 7.4) (Figure S2), which was significantly more stable than the
corresponding βꢀfucosyl fluoride (t1/2 ca. 20 min) 48,67. βꢀFucosyl azide (1) was used before as
substrate for testing α1,2ꢀ and α1,3ꢀfucosidase mutants 52. Thus, the potential glycosylation
activity of the nucleophilic mutants, including D200G, D200S, D200A, and D200T, were tested
using both the βꢀglycosyl azide (1) and the βꢀglycosyl fluoride (2) as the donor substrates and the
FmocꢀAsn(GlcNAc)ꢀOH (4) 68 as the acceptor substrate (Scheme 1). The reactions were run with
different amounts of enzymes and were monitored by reverseꢀphase HPLC analysis. However,
no glycosylation products were observed with either the βꢀfucosyl azide (1) or the
2ꢀdexoyꢀ2ꢀfluoroꢀβꢀfucosyl fluoride (2) donors by any of the nucleophilic residue mutants. This
result suggested that those nucleophilic mutants so far tested did act as a glycosynthase.
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Next, we tested the potential glycoꢀligase activity of the general acid/base residue mutants
using the αꢀfucosyl fluoride (3) as the donor substrate (Scheme 1). Interestingly, the E274A
mutant exhibited excellent catalytic activity to transfer a fucose residue to the GlcNAc moiety of
acceptor 4 to give the disaccharide derivative (5) with regioꢀ and stereoꢀspecificity (Scheme 1).
Using a moderate excess of the fucosyl fluoride (donor/acceptor, 2:1 molar equivalent), an
almost quantitative yield of product 5 was achieved by incubation of E274A (0.1 mg/ml, pH 7.5)
with 3 and 4 at 42 oC within 1 h. The other two mutants, E274G and E274S, were also excellent
catalysts for the α1,6ꢀfucosylation and gave similar results.
The structure of 5 was confirmed by ESIꢀMS (calculated, M = 703.3 Da; found, 704.5 [M +
1
H]+) and Hꢀ1H COSY NMR (Figure S3). A doublet at δ 4.62 with a relatively small coupling
constant (J = 3.2 Hz) for the Hꢀ1 of the fucose moiety suggested that the attached fucose was in
an αꢀglycosidic linkage. On the other hand, an obvious shift for the Hꢀ6 in the GlcNAc moiety of
5 toward the low field (from δ3.6 in 4 to δ3.8 in 5), while other protons on the GlcNAc moieties
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in 4 and 5 did not show obvious change, suggested that the fucosylation occurred at the
6ꢀhydroxy group of the GlcNAc moiety to form an α1,6ꢀfucosyl glycosidic linkage.
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Interestingly, we observed that in the absence of the GlcNAc acceptor, the AlfC mutants
(E274A, E274G, and E274S) only hydrolyzed αꢀfucosyl fluoride slowly but the wild type AlfC
could quickly hydrolyze the donor substrate. Taken together, these results indicated that the AlfC
mutants represented a class of unique Oꢀfucoligase for coreꢀfucosylation, which could use
synthetic αꢀfucosyl fluoride as the simple donor substrate instead of the expensive GDPꢀfucose
as the substrate as required by the α1,6ꢀfucosyltransferase (FUT8). To examine whether the
fused CPD domain in the enzyme played a role in catalyzing fucosylation, we removed the CPD
tag in the AlfC E274A mutant by using inositol hexaphosphate (Figure S4). The purified mutant
without CPD tag showed comparable catalytic activity as its CPD tagged counterpart, indicating
that CPD tag did not affect the catalytic activity of the mutant (Data not shown). However, we
found that the mutants fused with the CPD were more thermostable than the free enzyme. While
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incubation of the free enzyme at 55 C for 5 h led to partial precipitation probably due to
aggregation, the CPD fused mutant was still active, indicating that the CPD domain could
stabilize the enzyme. A detailed study on the fucosylation activity of E274A indicated that the
optimal pH of its catalysis was 7ꢀ8.5 and the optimal temperature was 40ꢀ50 oC (Figure S5).
We also performed kinetic studies on the mutants and the results were summarized in Table
S2 and Figure S6. The E274A mutant exhibited the highest fucosyl transfer activity with the
catalytic efficiency (kcat/KM) of up to 1.6 × 103 minꢀ1mMꢀ1. The E274S and E274G mutants also
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showed high activities (Table S2). In particular, kinetic analysis revealed that these mutants had
high turnover numbers (kcat > 200 minꢀ1) implying that these mutants, with mutation at the
general acid/base residue, could efficiently use the αꢀfucosyl fluoride for transglycosylation
without hydrolysis of the product.
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Direct coreꢀfucosylation of various GlcNAcꢀpeptides. The discovery that the E274A and
related mutants were able to efficiently catalyze the transꢀfucosylation on the GlcNAcꢀAsn
derivative prompted us to test if the mutants could also fucosylate GlcNAc moiety in the context
of polypeptides. We chose three distinct GlcNAcꢀcontaining peptides for the test: the
hexapeptide (6) derived from the sialylglycopeptide (SGP) isolated from egg yolks, a 19ꢀmer
GlcNAcꢀpeptide (8) that consisting of the CD52 antigen and a sortase A signal peptide sequence,
and a potent HIV inhibitor GlcNAcꢀC34 (10) derived from the HIVꢀ1 gp41 envelope
glycoprotein 69.
We found that the E274A could transfer a fucose moiety to all the three GlcNAcꢀpeptides to
form the corresponding fucosylated peptides (7, 9, and 11), respectively (Scheme 2). While the
small GlcNAcꢀpeptide (6) was transformed faster than the large ones (8 and 10), all the
enzymatic fucosylation could be achieved essentially quantitatively within a relatively short time
of incubation (1ꢀ2 h) when a sufficient amount of enzyme was used. The glycopeptide products
were readily purified and its identity was confirmed by LCꢀMS analysis (Figure S7). The
Fucα1,6GlcNAcꢀpeptides could serve as excellent acceptor substrates for the EndoꢀF3 D165A
glycosynthase to synthesize coreꢀfucosylated complex Nꢀglycopeptides 66.
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Scheme 2. Core fucosylation of GlcNAc-peptides by the fucoligase a)
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HO
OH
O
OH
HO
HO
HO
O
O
NH
O
HO
HO
NHAc
NH
O
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NHAc
K-L-A-N-K-T
O
K-L-A-N-K-T
6
7
OH
HO
OH
O
OH
O
OH
OH
HO
HO
HO
AcHN
O
F
3
O
NH
O
HO
HO
NH
AcHN
O
O
i)
quantitative
G-Q-N-D-T-S-Q-T-S-S-P-S-L-P-K-T-G-G-S
G-Q-N-D-T-S-Q-T-S-S-P-S-L-P-K-T-G-G-S
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HO
OH
OH
O
O
HO
O
HO
HO
O
NH
HO
HO
AcHN
NH
AcHN
O
O
Ac-W-M-E-W-D-R-E-I-N-N-Y-T-S-L-I-H-S-L-I-E-E-S-Q-N-Q-Q-E-K-N-E-Q-E-L-L
Ac-W-M-E-W-D-R-E-I-N-N-Y-T-S-L-I-H-S-L-I-E-E-S-Q-N-Q-Q-E-K-N-E-Q-E-L-L
10
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a) Reagents and conditions: i) 0.001-0.0015 mol. equiv. AlfC E274A, PBS (pH7.5), 42oC, 0.5-2 h
Direct core fucosylation of intact Nꢀglycopeptides. Next, we tested the feasibility of
E274Aꢀcatalyzed directed coreꢀfucosylation of intact Nꢀglycopeptides carrying a fullꢀsize
Nꢀglycan. Several complex Nꢀglycopeptides (12, 14, 16, and 18) carrying a sialylated
biantennary complex type Nꢀglycan were used as potential acceptor substrates, including a large
cyclic HIVꢀ1 V3 glycopeptide (18). Surprisingly, the E274 mutant could efficiently add a core
fucose to the intact Nꢀglycopeptides to give the corresponding coreꢀfucosylated Nꢀglycopeptides
(13, 15, 17, and 19), respectively in excellent yields (Scheme 3). The glycopeptide products were
separated by RPꢀHPLC and their identities were first characterized by ESIꢀMS analysis (Figures
S8).
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To verify that the fucose was specifically added to the innermost GlcNAc moiety in the
glycopeptide, the isolated glycopeptide product (15) was treated with two specific hydrolytic
enzymes PNGase F and EndoꢀF3 that hydrolyze Nꢀglycopeptides and Nꢀglycoproteins. PNGase
F completely removes the Nꢀglycans from Nꢀglycopeptides by hydrolyzing the amide linkage
between asparagine and the Nꢀglycans, while EndoꢀF3 specifically cleaves at the β1,4ꢀglycosidic
bond between the two GlcNAc moieties in Nꢀglycopeptides/proteins. ESIꢀMS analysis indicated
that treatment of 15 with PNGase F efficiently converted 15 into a single peptide species that
was corresponding to the free polypeptide without any sugar attached (Calcd for the plain
polypeptide, M = 1848.75 Da; found, M= 1848.83 Da) (Figure S9A), suggesting that the fucose
must be transferred to the Nꢀglycan portion instead of any residues on the peptide portion. It
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should be pointed out that enzyme PNGase F is highly sensitive to the site of fucosylation in the
N,Nꢀdiacetylchitobiose core, which could tolerate core fucosylation at the 6ꢀOH of the innermost
GlcNAc moiety, but could not release Nꢀglycans with an α1,3ꢀfucose being attached to the core,
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as usually found in plant Nꢀglycoproteins . The efficient release of the Nꢀglycans from the
Nꢀglycopeptide by PNGase F treatment further confirmed that the fucose moiety was α1,6
attached to the innermost GlcNAc moiety. On the other hand, treatment of 15 with EndoꢀF3
gave a species that corresponded to Fucα1,6GlcNAcꢀpeptide (9) (ESIꢀMS, calculated, M=
2195.38 Da; found, M = 2196.64 Da, deconvolution data) (Figure S9B). Taken together, all these
experimental data suggested that indeed the fucose moiety was added specifically to the
innermost, Asnꢀlinked GlcNAc moiety of the glycopeptide in the glycoligaseꢀcatalyzed
fucosylation. The ability to directly coreꢀfucosylated intact Nꢀglycopeptides opens a new avenue
to access coreꢀfucosylated Nꢀglycopeptides directly from natural and synthetic Nꢀglycopeptides.
Direct core fucosylation of intact glycoproteins. The success in direct coreꢀfucosylation
intact glycopeptides by the glycoligase mutants encouraged us to also test the direct
coreꢀfucosylation of intact Nꢀglycoproteins. Thus, we chose bovine ribonuclease B (RNase B,
20), a natural glycoprotein with heterogeneous highꢀmannose type Nꢀglycan, as a model system
to investigate whether this engineered enzyme was capable of fucosylating the natural
glycoprotein. RNase B is a good model for the study of protein glycoꢀremodeling, as it has only
a single Nꢀglycosylation site, N34, located around the cleft of the spheroidal protein. The
Nꢀglycans on natural RNase B are a mixture of high mannose type from five to nine mannose
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residues. Firstly, we examined whether the E274A mutant could perform direct coreꢀfucosylation
on the glycoform mixtures and, if yes, whether the mutant showed any selectivity on the
Nꢀglycans of different size. Thus, RNase B (20) was incubated with 3 and the E274A mutant at
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37 C (a lower temperature to prevent protein denaturation). The reaction was monitored with
LCꢀMS. To calculate the reaction yields, glycans were cleaved from the protein with PNGase F
and then detected and quantitated with HPLC after Fmocꢀlabelling (See experimental section) 71.
We found that RNase B (20) could serve as an acceptor substrate of AlfC E274A for
coreꢀfucosylation (Scheme 4), but the reaction was relatively slow, giving partial transformations
to the fucosylated RNase B glycoforms 21 under the conditions after 5 h (Figure S10).
Interestingly, the E274A mutant exhibited some substrate specificity on the different glycoforms.
The yields of core fucosylation of respective glycoforms, as judged by the HPLC analysis of the
released coreꢀfucosylated Nꢀglycans were 72%, 54%, 51%, 55% and 43%, for the Man5 to Man9
glycoforms, respectively (Figure S11). These results suggested that for highꢀmannose type
Nꢀglycoforms, the larger size Nꢀglycans were less favorable substrate for E274A, probably due
to steric hindrance. This study also demonstrated that highꢀmannose type glycoproteins could
serve as substrates for coreꢀfucosylation by the fucoligase mutant.
Since the direct coreꢀfucosylation of RNase B was less efficient, we tested an alternative
approach that consists of deglycosylation, glycoligaseꢀcatalyzed coreꢀfucosylation, and EndoꢀF3
catalyzed transglycosylation to constitute a coreꢀfucosylated complex type RNase glycoform
(Ribonuclease C) (Scheme 4). Thus, The high mannose Nꢀglycans in RNase B (20) were
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removed by EndoꢀH to afford the deglycosylated protein GlcNAcꢀRNase (22) in quantitative
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yield . We found that, in contrast to the slow fucosylation of the intact RNase B (20), the core
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fucosylation of GlcNAcꢀRNase (22) was much more efficient to give the
Fucα1,6GlcNAcꢀRNase (23) in essentially quantitative yield within 5 h under the same
conditions. Deconvolution of the ESIꢀMS spectrum gave a single species confirming the identity
of the fucosylated product (Calculated for 23, M = 14031 Da; found, M = 14034 Da) (Figure
S12A). Subsequently, EndoꢀF3 D165A catalyzed glycosylation of the Fucα1,6GlcNAcꢀRNase
(23) with a complex glycan oxazoline (24), following our previously reported procedure 66, gave
the core fucosylated complex glycoform (25) in 97% yield (Scheme 4). ESIꢀMS analysis verified
the glycosylation product (Calculated for 25, M = 16033 Da; found, M = 16036 Da) (Figure
S12B). This was the first synthesis of coreꢀfucosylated complex type glycoform of RNase
(RNase C). The synthesis was more efficient than the preparation of the nonꢀfucosylated RNase
C that we have previously reported using EndoꢀM mutant for transglycosylation 73. The ability of
the fucoligase mutant to directly fucosylate intact glycopeptides and glycoproteins significantly
expands the repertoire of toolkits for making homogeneous core fucosylated glycoforms for
various glycomic applications.
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Direct enzymatic coreꢀfucosylation of intact monoclonal therapeutic antibody. The
potent transfucosylation activity of AlfC αꢀfucoligase towards glycopeptides and glycoproteins
prompted us to explore its application for glycoengineering of therapeutic monoclonal antibodies.
Core fucosylation of the Fc domain Nꢀglycans plays a pivotal role in modulating the functions of
antibodies and their therapeutic efficacy 8,74. We have previously developed a chemoenzymatic
Fc glycan remodeling method that consists of deglycosylation and glycosynthaseꢀcatalyzed
glycan transfer to produce homogeneous Fc glycoforms 65,66,75. If the fucoligase mutant could act
on intact antibody, then it would further expand the repertoire of the toolbox for antibody Fc
glycan engineering. To test this possibility, we selected rituximab (26) as a model system. First,
rituximab was deglycosylated with EndoꢀS followed by defucosylation with the wild type AlfC
αꢀfucosidase to give the GlcNAcꢀrituximab (27) (Scheme 5). Interestingly, Wildꢀtype AlfC
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αꢀfucosidase showed good hydrolytic activity, to afford the deglycosylated product (27) with
essentially complete transformation within 3 h. However, we found that Wildꢀtype AlfC
αꢀfucosidase was unable to remove the fucose moiety in the intact rituximab before EndoꢀS
treatment, even with significantly large amount of the enzyme and prolonged incubation time
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(data not shown). The nonꢀfucosylated sialyl complex type glycoform
of rituximab
(S2G2ꢀrituximab, 28) was synthesized by the EndoSꢀD233Q catalyzed glycosylation with glycan
oxazoline (24) as the donor, following our previously published procedure 75. These two
glycoforms (27 and 28) were used as potential substrates for examining the AlfC fucoligase
mutants. The GlcNAcꢀrituximab (27) was incubated with αꢀfucosyl fluoride (3) and AlfC E274A
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mutant at 37 C and the reaction was monitored by LCꢀMS analysis. We found that
GlcNAcꢀrituximab acted as an excellent substrate of the fucoligase AlfC E274A and the
enzymatic reaction resulted in complete coreꢀfucosylation of 27 to give the
Fucα1,6GlcNAcꢀrituximab (29) as a single product within 6 h under the conditions. The product
was readily isolated by protein A affinity chromatography. To confirm the identity of the
Fucα1,6GlcNAcꢀrituximab (29), the product was treated with protease IdeS which specifically
hydrolyzes IgG antibody to release the monomeric Fc domain, and the released Fc was analyzed
by ESIꢀMS. The deconvolution of the ESIꢀMS of core fucosylated Fc domain exhibited a single
species with the expected molecular mass (Calculated for Fucα1,6GlcNAcꢀFc, M = 24112 Da;
found, M = 24110 Da) (Figure S13). The 146 Da increase from 27 to 29 suggested an addition of
a single fucose moiety in the monomeric Fc domain. As described in our previous study,
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Fucα1,6GlcNAcꢀrituximab (29) was an excellent substrate for EndoSꢀD233Q 75. Indeed,
incubation of 29 with EndoSꢀD233Q and glycan oxazoline (24) gave the expected
coreꢀfucosylated sialyl complex type glycoform of rituximab (S2G2Fꢀrituximab, 30) in excellent
yield, further confirming that the 29 was the expected Fucα1,6GlcNAcꢀrituximab.
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Finally, we tested the direct fucosylation of S2G2ꢀrituximab (28) carrying the full size
complex type Fc Nꢀglycans. Interestingly, we found that the AlfC E274 mutant could also
smoothly transfer the fucose moiety to the intact rituximab, albeit at a slow rate in comparison
with the GlcNAcꢀrituximab substrate. Nevertheless, an increased amount of the enzyme could
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lead to complete transformation of the S2G2ꢀrituximab (28) to the coreꢀfucosylated glycoform
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(S2G2Fꢀrituximab, 30) within a few hours at 37 C (Scheme 5). This result is remarkable given
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the fact that the Fc domain Nꢀglycans are buried between two Fc domains, and that wild type
AlfC αꢀfucosidase actually shows almost no activity to remove the core fucose in the intact Fc
Nꢀglycans. It is puzzling why the wild type αꢀfucosidase was unable to hydrolyze the fucose in
the intact antibody but the E274A mutant could manage to access the site to efficiently perform
the transꢀfucosylation on the intact antibody.
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To confirm that the fucose was specifically attached at the core position and that there was
no nonꢀenzymatic attachment of the fucose to any residues in the protein portions, we performed
ESIꢀMS analysis of the intact antibody product, coupled with specific enzymatic transformations
(Figure 3). First, the observed molecular mass of 30 from the ESIꢀMS deconvolution data
matched well the expected molecular mass (Calcd, M = 148888 Da, found, M = 148886 Da)
(Figure 3A). The data suggested an addition of two fucose moieties on top of the
S2G2ꢀrituximab (28) that was a dimer carrying two Nꢀglycans (each heavy chain has an
Nꢀglycan at the Asnꢀ297 site); second, PNGase F treatment of 30 gave a single species of the
antibody, the ESIꢀMS deconvolution data (144182 Da) of which agreed with the polypeptide
backbone of rituximab (calcd, M = 144181 Da) (Figure 3B). This result suggested that there was
no any additional modification of the protein portions of rituximab during the AlfC E274A
catalyzed reactions; Third, EndoꢀS treatment of 30 converted it back to the
Fucα1,6GlcNAcꢀrituximab (29), which appeared as a single species at 144882 Da (Figure 3C).
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Lectin binding studies with coreꢀfucosylated RNase B and rituximab. The interactions of
glycoproteins and core fucoseꢀspecific lectins play important roles in biological recognition
processes. To demonstrate the specific recognition, we performed the binding analysis of the
interactions between the Aleuria aurantia lectin (AAL) and a few coreꢀfucosylated glycoproteins
synthesized via the AlfC fucoligase catalyzed reactions. AAL is a commonly used lectin, which
has high affinity for α1,6ꢀfucosylated glycoproteins, including core fucosylated antibody and
haptoglobin 76,77. First we tested the binding of 21 carrying core fucosylated highꢀmannose
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glycans and the natural glycoprotein (20) with AAL. The AAL showed specific binding to 21,
while it didn’t show any binding to the nonꢀfucosylated glycoprotein (20) (Figure 4A). AAL
also demonstrated specific recognition of the S2G2Fꢀrituximab (30) but did not show any
detectable affinity to the S2G2ꢀrituximab (28) (Figure 4B). Interestingly, a significant difference
in the affinities of 21 and 30 for lectin AAL was observed (EC50: 275 ꢀM for 21 vs. 15 ꢀM for
30), with the affinity of the coreꢀfucosylated antibody being about 17 fold higher than that of
coreꢀfucosylated RNase B. To verify whether the observed difference majorly came from the
difference in the nature of the Nꢀglycans (core fucosylated highꢀmannose type in 21 vs. complex
type in 30) or in the nature of proteins (the context of RNase vs. antibody), we released the
Nꢀglycans from the proteins by PNGase F treatment with in situ Fmoc labeling 71. ELISA
analysis of the AAL binding indicated that the affinity of the coreꢀfucosylated complex type
Nꢀglycans (EC50, 1.8 ꢀM) released from antibody (30) was about 8 fold higher than that of the
coreꢀfucosylated highꢀmannose type Nꢀglycans (EC50, 16 ꢀM) released from (21). Thus, the
difference in AAL affinities between coreꢀfucosylated RNase B (21) and coreꢀfucosylated
antibody (30) came mainly from the difference in the nature of the Nꢀglycans (high mannose vs.
complex type), and the protein part contributed to a less extent (ca. 2ꢀfold).
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S2G2ꢀrituximab (28)
S2G2Fꢀrituximab (30)
Intact RNase B (20)
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Fucosylated RNase B (21)
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0.1
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Concentration of AALꢀbiotin (
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Concentration of AALꢀbiotin (ꢀM)
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Free glycan released from 28
Free fucosylated glycan from 30
Free glycans released from 20
Free fucosylated gylcan from 21
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Concentration of AALꢀbiotin (
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Figure 4. ELISA analysis of the binding between lectin AAL and glycoproteins or released Nꢀglycans. The glycoproteins or
released Nꢀglycans (tagged with Fmoc) were immobilized on the plates and probed by serial dilutions of the lectin. A) AAL
binding with RNase B (20) and fucosylated RNase B (21); B) AAL binding with nonꢀfucosylated glycoform of rituximab (28)
and fucosylated rituximab glycoform (30); C) AAL binding with the PNGase F released highꢀmannose Nꢀglycans released
from 20 and 21; D) AAL binding with the PNGase F released complex type Nꢀglycans from 28 and 30.
Comparative SPR binding studies with coreꢀfucosylated and nonꢀfucosylated
glycoforms of antibody rituximab. Previous studies have demonstrated that core fucosylation
adversely impacts the binding of IgG antibodies to the FcγIIIA receptor (FcγRIIIA) on natural
killer cells and thus downꢀregulates the antibodyꢀdependent cellular cytotoxicity (ADCC) 74,78,79
.
To verify the effects of fucosylation on Fc receptor binding of the coreꢀfucosylated rituximab
glycoforms obtained through the fucoligase technology, we performed the surface plasmon
resonance (SPR) analysis on the binding of the FcγRIIIAꢀV158 (a major highꢀaffinity FcγIIIA
receptor allele) to the nonꢀfucosylated glycoforms (27 and 29) vs. the corresponding
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coreꢀfucosylated glycoforms (28 and 30) (Figure 5). The rituximab glycoforms were
siteꢀspecifically immobilized on protein A chips and the FcγRIIIAꢀV158 at various
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concentrations were injected as analytes, following our previously reported procedures . As
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expected, the nonꢀfucosylated sialyl complex type glycoform (S2G2ꢀrituximab, 28) showed
much higher affinity than that of the corresponding coreꢀfucosylated glycoform
(S2G2Fꢀrituximab, 30). As estimated by the dissociation constant (KD), the affinity of
S2G2ꢀrituximab (28) (KD = 2.2 nM) for FcγRIIIAꢀV158 was about 56ꢀfold higher than that of
the corresponding fucosylated S2G2F glycoform (30) (KD = 131 nM) (Figure 5A and 5B). This
result was consistent with our previously reported binding data 74. On the other hand, the
deglycosylated glycoforms (27 and 29) showed much lower affinity than that of the glycosylated
glycoforms (Figure 5C and 5D). Even in this case, the effects of coreꢀfucosylation was apparent.
While the GlcNAcꢀrituximab (27) carrying only the innermost GlcNAc moiety at the Fc domain
showed a moderate affinity for the FcγIIIA receptor with a KD of 784 nM, the corresponding
coreꢀfucosylated glycoform, Fucα1,6GlcNAcꢀrituximab (29), demonstrated only marginal
binding to the receptor under the measurement condition, for which the KD was not determined.
These data confirm that core fucosylation could remarkably reduce the affinity of IgG antibodies
for the activation receptor (FcγIIIA), which would significantly decrease antibodyꢀdependent
cellular cytotoxicity, as demonstrated in clinical trials 79. On the other hand, a decreased affinity
for the activation Fc receptor could be beneficial if the antibody would be used for the treatment
of autoimmune diseases, as demonstrated by the Fc sialylationꢀenriched intravenous
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immunoglobulin (IVIG) that showed significantly enhanced antiꢀinflammatory activity in a
mouse model of rheumatoid arthritis (RA) 80,81. Thus, the fucoligaseꢀcatalyzed direct
coreꢀfucosylation of intact antibodies provides an efficiency avenue to modulating antibody’s
immunological properties.
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CONCLUSION
A highly efficient chemoenzymatic method for direct coreꢀfucosylation of intact
Nꢀglycopeptides, Nꢀglycoproteins, and therapeutic antibodies is established. This method was
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enabled by the discovery of an array of AlfC αꢀfucosidase mutants that act as novel glycoligases
for transglycosylation using αꢀfucosyl fluoride as the simple donor substrates. The AlfC
αꢀfucosidase mutants represent the first examples of glycoligases capable of specifically
attaching an α1,6ꢀfucose moiety to intact Nꢀglycans of glycoproteins. The discovery of the novel
αꢀfucoligases opens a new avenue to quickly constructing library of coreꢀfucosylated
Nꢀglycopeptides and Nꢀglycoproteins directly from the corresponding nonꢀfucosylated
counterparts, which have been hitherto difficult to obtain for various glycomics studies.
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EXPERIMENTAL SECTION
Materials and Methods
All chemicals, reagents and solvents were purchased from Sigma–Aldrich and TCI, and
unless specially noted, applied in the reaction without further purification. Monoclonal antibody
rituximab was purchased from Premium Health Services Inc. (Columbia, MD). Silica gel (200–
425 mesh) for flash chromatography was purchased from Sigma–Aldrich. Analytical
reverseꢀphase chromatography was performed on a Waters 626 HPLC instrument equipped with
an XBridge BEH130 C18 column (3.5 ꢁm, 4.6 × 250 mm) for reversed phase or YMCꢀPack NH2
column (5 ꢁm, 4.6 × 250 mm) for normal phase. The XBridge column was eluted with a linear
gradient of acetonitrile (0ꢀ30%, v/v) with water containing TFA (0.1%) over 35 min at a flowrate
of 0.5 mL/min under UV 214nm. The YMCꢀPack NH2 column was eluted with a linear gradient
of ammonium formate (100mM, pH4.5, 10ꢀ60%, v/v) with acetonitrile containing TFA (0.1%)
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over 80 min at a flowrate of 0.5 mL/min under UV 266nm. Preparative HPLC was performed on
a Waters 600 HPLC instrument equipped with a SymmetryPrepTM C18 column (7 ꢁm, 19 × 300
mm). HighꢀPerformance AnionꢀExchange Chromatography with Pulsed Amperometric Detection
(HPAECꢀPAD) was performed on a Dionex ICSꢀ5000 chromatography system (Fischer
Scientific) equipped with an electrochemical detector (ED50) and an anion exchange column
(CarboPac PA10 4 × 250 mm). The PA10 column was eluted with a constant buffer composed of
80% 100 mM NaOH and 20% water at a flowrate 1.0 mL/min. Liquid Chromatography
Electrospray Mass spectrometry (LCꢀESIꢀMS) was used to analyze transfer products including
core fucosylated glycopeptides and glycoproteins. The LCꢀESIꢀMS was performed on an
Exactive™ Plus Orbitrap Mass Spectrometer (Thermo Scientific) equipped with a C18 column
(proZap Expedite MS C18, 2.1 × 10 mm, 1.5 ꢁm, P.J. Cobert Associates, Inc.) for glycopeptides,
C8 column (Poroshell 300SBꢀC8, 1.0 × 75 mm, 5 ꢁm, Agilent) for glycoproteins and Cꢀ4
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column (XBridgeTM BEH300 C4, 2.1 × 50 mm, 3.5 ꢁm, Waters) for antibody analysis. H, C
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and Hꢀ1H COSY spectra were recorded on a 400 or 600 MHz spectrometer (Bruker, Tokyo,
Japan) with D2O or DMSOꢀd6 as the solvent. Amino acid sequence alignment for different
fucosidases was performed using MAFFT, a multiple sequence alignment tool. Plasmid pCPD
vector (CPDꢀLasso) was created by engineering the CPD protein as a Cꢀterminal tag into
pET31(b) (Novagen) vector. PfuUltra II fusion HS DNA polymerase was purchased from Agilent.
Restriction enzymes and Escherichia coli competent cells including 5ꢀalpha and BL21 (DE3),
used for DNA manipulation and protein production, were purchased from New England BioLabs
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