An endoglycosidase with alternative glycan specificity allows broadened glycoprotein remodelling

Article abstract of DOI:10.1021/ja301334b

Protein endoglycosidases are useful for biocatalytic alteration of glycans on protein surfaces, but the currently limited selectivity of endoglycosidases has prevented effective manipulation of certain N-linked glycans widely found in nature. Here we reve

Full text of DOI:10.1021/ja301334b

Communication
pubs.acs.org/JACS
An Endoglycosidase with Alternative Glycan Specificity Allows
Broadened Glycoprotein Remodelling
Jonathan J. Goodfellow,†,§ Kavitha Baruah,‡,§ Keisuke Yamamoto,†,§ Camille Bonomelli,‡
Benjamin Krishna,‡ David J. Harvey,‡ Max Crispin,‡ Christopher N. Scanlan,*,‡ and Benjamin G. Davis*,†
†Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
‡Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
S
* Supporting Information
ABSTRACT: Protein endoglycosidases are useful for
biocatalytic alteration of glycans on protein surfaces, but
the currently limited selectivity of endoglycosidases has
prevented effective manipulation of certain N-linked
glycans widely found in nature. Here we reveal that a
bacterial endoglycosidase from Streptococcus pyogenes,
EndoS, is complementary to other known endoglycosi-
dases (EndoA, EndoH) used for current protein
remodeling. It allows processing of complex-type N-linked
glycans +/− core fucosylation but does not process
oligomannose- or hybrid-type glycans. This biocatalytic
activity now addresses previously refractory antibody
glycoforms.
lycan display is critical for development and physiology of
G
many living systems.1 Protein glycosylation is a diverse
modification comprising 50% of cellular proteome and 90% of
secreted proteome.2 Of proteins currently in clinical trials, 70%
are glycoproteins,3 typically with “core fucosylation” (Figure 1).
Natural glycoproteins exist as mixtures of glycoformssame
peptide backbone but different glycosylation pattern and site
making isolation of well-defined glycoproteins complicated.4
Since certain glycoforms are more active,5 access to specific
uniform types can confer therapeutic advantages. In the case of
antibodies (Abs), altering IgG-Fc glycosylation modulates
binding to cellular receptors, thus tuning cytoxicity6 and
determining anti-inflammatory activity.7
Figure 1. (a) N-linked glycoprotein biosynthesis and potential
corresponding methods (magenta) to access altered glycans.14 (b)
Glycan remodeling using Endo enzymes (here idealized complex-type;
key from ref 15). (c) Trimming of human IgG glycans by EndoS, not
EndoA/H. MALDI-TOF MS ([M + Na]+) confirms unique complex-
type processing (see also Figure S8).
Recombinant expression systems and chemoenzymatic and
chemical methods can supply some homogeneous synthetic
glycoproteins.8 One powerful approach is enzymatic remodel-
ing: initial heterogeneous glycoform mixtures are treated with
an endoglycosidase (“Endo”) to trim off the variable portions of
the oligosaccharides attached to the first N-acetylglucosamine
(GlcNAc) residue at N-linked site(s) (Figure 1b). Subsequent
enzyme-mediated glycosylation of the exposed GlcNAc residue
can then produce a more homogeneous sample.8d,9 However,
Endo enzymes currently used to attach glycans to proteins in
this way show restricted sugar selectivity that prevents such
combined removal and attachment of the complex-type glycans
normally found on mammalian proteins or glycans that contain
“core fucose”.10−12
commonly used in current synthesis, orfs exist in other families,
implying possibly different substrate specificities. The ndoS
gene from Streptococcus pyogenes (GH18) codes for production
of an apparently immunomodulatory evasion factor that
prolongs bacterial survival and acts upon four subclasses of
IgG.19,20 Codon-optimized ndoS gene was generated (Table
S1) and subligated into pGEX-4T-1 vector, which allowed
efficient (∼10 mg/L) heterologous expression of EndoS in N-
terminally-GST-tagged form using Escherichia coli BL21(DE3).
To identify new activities, we surveyed known and putative
Endo (E.C. 3.2.1.96) orfs (putative ndo genes). While those in
the CAZy glycoside13 hydrolase family GH85 are most
Received: February 22, 2012
Published: May 2, 2012
© 2012 American Chemical Society
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Communication
Figure 2. EndoS digestion of IgG glycans. ESI(−)-MS of human serum IgG N-linked glycans released by (a) PNGase F, (b) EndoS, and (c) PNGase
F after EndoS. Inset in (c): ESI(−)-MS/MS of (i) m/z 1762.6 and (ii) m/z 1924.6. Fragment ions are labeled according to the scheme proposed by
Domon and Costello;16 structural identification is as described by Harvey.17,18
Figure 3. EndoS digestion of free N-linked glycans from desialylated recombinant gp120BaL. MALDI(+)-MS spectra (a) of total gp120 glycan pool
and (b) following digestion with EndoS. The red lines highlight the cleaved species and their products.
A thrombin-cleavable site allowed EndoS to be produced
tagged and untagged (Table S2); both were highly active.
To evaluate important targets with relevant glycosylation, we
restricted analyses to glycoprotein substrates from mammalian
expression. Human IgG (Figures S7 and S8) was incubated
with EndoS or widely used EndoA (GH85)21 and EndoH
(GH18)22 at 37 °C for 1 h in PBS (pH 7.4, 10 mM). Reducing
SDS-PAGE and MALDI(+)-MS analyses revealed absence of
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Journal of the American Chemical Society
Communication
Figure 4. Alternative processing of N-glycans (PNGase-F-released) with distributions from modulated glycosylation pathways. Both hybrid (left)
and oligomannose Man9GlcNAc2 (+ Man5−8GlcNAc2) (right) were processed by EndoH, not EndoS.
activity by EndoA and EndoH toward IgG. In contrast, EndoS
processed IgG efficiently; glycans were removed even at low
concentration (<50 nM, kapp > 0.8 s−1). Detailed analyis by
ESI(−)-MS (Figure 2) revealed clean release of G0, G1, and
G2 glycans (including sialylated variants) by EndoS. Only a
trace population (<2%) of bisected biantennary structures was
found to be resistant to exhaustive digestion (consistent with
tryptic mapping23); composition was confirmed by ESI-MS/
MS (Figure 2c). The high activity of EndoS against IgG glycans
contrasted starkly with lack of cleavage of these sugars by
EndoA and EndoH (Figure S8).
1b) Abs produced in mammalian eukaryotic systems and hence
many current therapeutics. To test this promise, we remodeled
heterogeneous glycoforms of IgG (Figure 5). Endo-β-N-
Having confirmed EndoS’s atypical ability to process IgG
glycans, we tested it against those from other important
recombinant glycoproteins. We used HIV-1 glycoprotein,
gp120, with a wide range of complex, hybrid, and
oligomannose-type glycans when expressed from human
embryotic kidney (HEK293T) cells. Analysis of free gp120
N-glycans confirmed susceptibility of complex, fucosylated N-
linked carbohydrates to EndoS (Figure 3). Bisected structures
were not cleaved; additional EndoS-resistant glycoforms were
identified (e.g., triantennary/oligomannose-type). These alter-
native specificities of EndoS and EndoH were assayed by
expression of recombinant IgG-Fc with oligomannose or
hybrid-type glycans (Figure 4), derived through inhibitory
modulation of HEK293T glycosylation (Figure 1a, SI). Both
were deglycosylated by EndoH but not EndoS, confirming
unique and alternative specificity. Thus, protein glycans
displayed strikingly different behaviors: EndoH cleaved only
oligomannose or hybrid structures, which were untouched by
EndoS. Conversely, in the presence of a wide range of N-linked
glycans, EndoS was uniquely capable of processing complex-
type glycans usually found on Abs, presenting a new route to
remodeling and synthesis.
Figure 5. Remodeling of human IgG glycans. (a) EndoS-catalyzed
glycosylation with 1 creates unnatural glycoform. (b) Native and (c)
remodeled PNGase-cleaved glycan MALDI-MS.
acetylglucosaminidases hydrolyze the glycosidic bond in the
N,N′-diacetylchitobiose core of N-linked glycans. Some, e.g.,
EndoA24 and EndoM,25 also possess significant transglycosy-
lation activity and have been used to remodel the N-
glycoprotein RNase-B.26 Importantly, the efficiency of glyco-
sylation has been improved27 using sugar oxazolines as
substrates and mutant Endo enzymes. Recently, re-engineering
of bacterial glycosylation9d or chemical attachment28 has
allowed repositioning of glycosylation site. Notably, previous
remodeling of yeast-produced Fc’s proved possible due to non-
fucosylated, high/oligo-mannose glycoform content.29 More-
over, other Endo approaches have proven difficult: a system
based on innovative adaptation of prokaryotic production gave
Fc yields of <5%.9d
Next, we tested EndoS’s processing of other glycan types.
Core fucosylation modulates effector function6 and is prevalent
in human and therapeutic Abs. IgG-Fc was transiently
expressed (see SI) in wild-type human (HEK293T) cells.
Processing of glycans in fucosylated, non-fucosylated, and
mixed (Figure S9a−c) states by EndoS revealed valuable
plasticity for “core” α(1,6)-Fuc. This demonstrated that EndoS
has, by virtue of its selectivity, the potential to remodel (Figure
Tetrasaccharide oxazoline 1 was synthesized (SI) and after
trimming was used in EndoS-catalyzed glycosylation of IgG to
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Communication
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ASSOCIATED CONTENT
* Supporting Information
Protocols and further analysis. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
Chris.Scanlan@bioch.ox.ac.uk; Ben.Davis@chem.ox.ac.uk
■
Author Contributions
(25) Yamamoto, K. J.; Kadowaki, S.; Watanabe, J.; Kumagai, H.
Biochem. Biophys. Res. Commun. 1994, 203, 244.
§J.J.G., K.B., and K.Y. contributed equally.
(26) (a) Fujita, K.; Yamamoto, K. Biochim. Biophys. Acta 2006, 1760,
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Notes
The authors declare no competing financial interest.
(27) (a) Li, B.; Song, H.; Hauser, S.; Wang, L.-X. Org. Lett. 2006, 8,
3081. (b) Heidecke, C. D.; Ling, Z.; Bruce, N. C.; Moir, J. W. B.;
Parsons, T. B.; Fairbanks, A. J. ChemBioChem 2008, 9, 2045. (c) Rising,
T. W.; Heidecke, C. D.; Moir, J. W.; Ling, Z.; Fairbanks, A. J. Chem.
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ACKNOWLEDGMENTS
■
B.G.D. thanks BBSRC, EPSRC, Glycoform Ltd (J.J.G.), and
VTU (K.Y.) for funding and Drs. C. Urch, T. Purkarthofer, and
R. Weis for discussions. C.N.S. is funded by IAVI (no.
UOXFORCOA1101; Shimazu AXIMA equipment grant); K.B.
and C.B. by Glycobiology Institute scholarships.
(28) Fernandez-Gonzalez, M.; Boutureira, O.; Bernardes, G. J. L.;
Chalker, J. M.; Young, M. A.; Errey, J. C.; Davis, B. G. Chem. Sci. 2010,
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