Design of Artificial Glycosidases: Metallopeptides that Remove H Antigen from Human Erythrocytes

Article abstract of DOI:10.1002/anie.201612079

Catalysts that promote carbohydrate degradation have a wide range of potential applications, but the use of either enzyme glycosidases or small-molecule catalysts in biological systems raises significant challenges. Herein, we demonstrate a novel strategy for the design of synthetic agents that mimic natural glycosidases and address current problems for biological use. This strategy is illustrated by application to the development of potential blood substitutes for the rare Bombay blood type that is characterized by a deficiency of H2 antigen. Metallopeptides with 16 to 20 amino acids were constructed as artificial fucosidases that exhibit selective carbohydrate cleavage reactivity toward l-fucose over d-glucose. Selective fucose cleavage from the H2-antigen saccharide enables efficient removal of H2 antigen from erythrocytes and thereby accomplishes the conversion of regular human type-O blood into a potential blood substitute for the rare Bombay blood type.

Full text of DOI:10.1002/anie.201612079

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201612079
German Edition: DOI: 10.1002/ange.201612079
Artificial Enzymes
Design of Artificial Glycosidases: Metallopeptides that Remove
H Antigen from Human Erythrocytes
Zhen Yu and James Allan Cowan*
Abstract: Catalysts that promote carbohydrate degradation
have a wide range of potential applications, but the use of either
enzyme glycosidases or small-molecule catalysts in biological
systems raises significant challenges. Herein, we demonstrate
a novel strategy for the design of synthetic agents that mimic
natural glycosidases and address current problems for biolog-
ical use. This strategy is illustrated by application to the
development of potential blood substitutes for the rare Bombay
blood type that is characterized by a deficiency of H2 antigen.
Metallopeptides with 16 to 20 amino acids were constructed as
artificial fucosidases that exhibit selective carbohydrate cleav-
age reactivity toward l-fucose over d-glucose. Selective fucose
cleavage from the H2-antigen saccharide enables efficient
removal of H2 antigen from erythrocytes and thereby accom-
plishes the conversion of regular human type-O blood into
a potential blood substitute for the rare Bombay blood type.
trolled and targeted manner. The design of our artificial
glycosidases includes a targeting moiety that directs binding
toward the substrate of interest, and an independent carbo-
hydrate-degrading moiety that mediates oxidative chemistry
at the substrates under physiological conditions. Isolation of
the catalytic domain from the binding domain ensures greater
flexibility for the catalytic apparatus and improved selectivity
of the artificial glycosidases toward a broader range of
substrates containing the target sugar.
By applying this strategy, we designed artificial fucosi-
dases to produce a potential blood substitute for a rare blood
type. While recent studies have described protein-based
glycosidases that convert type-A blood and type-B blood
into universal type-O blood, there are few efforts to develop
blood substitutes for rare blood types owing to a lack of
[7]
economic incentives. Blood transfusions for patients with
rare blood groups are perceived to be a significant challenge
in transfusion medicine owing to the scarce resource of
T
he development of tools for the catalytic degradation of
carbohydrates has a broad range of potential applications.
[1]
[8]
donated blood from rare blood groups. Type-2 H antigen
Glycosidases are enzymes responsible for the selective
degradation of carbohydrates, however, their selectivity
toward specific carbohydrate substrates can be disadvanta-
(H2, or CD173) is the terminal polysaccharide residue of
glycolipids or glycoproteins on erythrocytes (red blood cells)
with the sequence Fuca1-2Galb1-4GlcNAcb. Deficiency of
[9]
[
2]
geous in the context of biotechnology application. The
presence of certain residues adjacent to the target carbohy-
drate can severely influence or even suppress enzyme
H2 antigen on the erythrocyte surface is known as the
[
9,10]
Bombay phenotype (also Oh, or h/h).
this phenotype is low (1 of 10000 in India, and 1 per million in
Europe).
The prevalence of
[
3]
[10,11]
activity. For example, an a-fucosidase from bacteroides
thetaiotaomicron can hydrolyze pNP-fucoside, but exhibits no
activity towards other fucose-containing disaccharides or
Serum in patients carrying the Bombay pheno-
type contains antibodies against the H2 antigen, therefore,
transfusion of regular type-O blood to patients with the
Bombay phenotype can lead to a severe and fatal transfusion
[
4]
trisaccharides. Additional problems for protein-based gly-
cosidases include limited options in terms of natural protein
families, many of which can cause immune responses in vivo,
[
12]
reaction. Herein, synthetic metallopeptides were designed
as artificial fucosidases to remove fucose from H2 antigens on
human erythrocytes and thereby produce a substitute for the
Bombay blood type.
[5]
and a requirement for non-neutral pH. As an alternative,
both small-molecule catalysts and heterogeneous catalysts
have been developed to promote the hydrolysis of carbohy-
drates, however, harsh conditions that involve non-neutral
pH, the use of organic solvents, and negligible discrimination
between different carbohydrate substrates undermine their
The cleavage domain, composed of an amino terminal
II
II
Cu - and Ni -binding (ATCUN) motif (GGH) was incorpo-
rated into a fucose-selective binding domain derived from
odorranalectin (OL: YASPKCFRYPNGVLACT) or its trun-
[
6]
[13]
potential application in biological systems. Herein, we
demonstrate a strategy to design synthetic agents as selective
artificial glycosidases that degrade carbohydrates in a con-
cated form (tOL: KCFRYPNGVLACT). In addition, two
C-terminal-amidated analogues were also designed, since
reduction of the negative charge may enhance the interaction
between the peptides and the negatively-charged surface of
[
14]
erythrocytes. The l-fucose binding affinity of the designed
[*] Z. Yu, Dr. J. A. Cowan
peptides was studied by isothermal titration calorimetry
Department of Chemistry and Biochemistry
The Ohio State University
(
ITC). CuGGH-tOL-NH displays significant binding affinity
2
for l-fucose, with a KD of 57.8 mm (Figure S3), which is
100 West 18th Avenue, Columbus, OH 43210 (USA)
consistent with the reported K (54.7 mm) of odorranalec-
D
E-mail: cowan@chemistry.ohio-state.edu
[13]
tin. Interestingly, the binding of the copper-free GGH-tOL-
Supporting information (including experimental details) and the
ORCID identification number(s) for the author(s) of this article can
be found under:
NH to l-fucose exhibits a negative DH in contrast to the
2
endothermic binding pattern of its copper-bound analogue
http://dx.doi.org/10.1002/anie.201612079.
(Figure S3). This change in DH can be ascribed to the
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1
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II
potential coordination to Cu by hydroxy groups from fucose,
for the selective l-fucose cleavage activity of these artificial
fucosidases.
[15]
with binding reflecting the entropic loss of water molecules.
Nevertheless, titration of l-fucose into other peptides, either
in copper-bound form or copper-free form, resulted in
a negligible enthalpy response that precluded calorimetric
evaluation of binding affinity. Surface plasmon resonance was
also used to measure the fucose binding affinity of all of the
peptides (Table 1 and Figure S4 in the Supporting Informa-
tion). In fact, removal of the first four residues (YASP) from
full-length odorranalectin improves the fucose-binding affin-
Some protein-based fucosidases have been discovered to
induce cleavage of pNP-fucose, while lower enzyme activity
was observed with polysaccharides as a result of their
[
4,19]
substrate selectivity.
To confirm the fucose cleavage
reactivity of our artificial fucosidases towards polysaccha-
rides, a trisaccharide Fuca1-2Galb1-4GlcNAc (H-trisacchar-
ide, exact mass = 529.2) was used as a model to represent the
H2 antigens of human erythrocyte (Figure 1a). The cleavage
products were separated from the intact trisaccharide by
Table 1: Binding affinity of the metallopeptides with l-fucose.
using an amino (NH ) column and then analyzed by mass
2
spectrometry (MS). CuGGH-tOL-NH2 exhibits efficient
cleavage of fucose from H-trisaccharide in the presence of
ascorbate and peroxide, where the diminishing MS response
of H-trisaccharide indicates its disappearance (Figure 1b). In
contrast to protein-based fucosidases, our artificial fucosi-
dases can display cleavage reactivity towards a more diverse
group of fucose substrates by virtue of their smaller size and
distinct mode of action. Specifically, they can overcome the
incomplete-cleavage issue that is often encountered with
CuGGH-
tOL-NH2
CuGGH-
tOL-OH
CuGGH-
OL-NH2
CuGGH-
OL-OH
[
a]
KD [mm]
61.3Æ7.5
105Æ17
77.6Æ6.9
124Æ7
[
a] Dissociation constants were measured by surface plasmon reso-
nance.
ity of peptides by around 18–26%, while amidation of the
C terminus improves the binding affinity by around 60–71%.
Therefore, the fucose-binding affinity mainly arises from the
domain K5–T17 of odorranalectin, while the N-terminal
YASP residues and the C-terminal carboxylate contribute
little to carbohydrate binding affinity.
[
20]
protein-based fucosidases.
from H-trisaccharide,
Following removal of fucose
disaccharide product (Galb1-
a
4GlcNAc, exact mass = 383.1) was identified by MS that
corresponds to the saccharide antigen of the Bombay
The carbohydrate-cleavage reactivity of the metallopep-
tides was evaluated by using chromogenic substrates linked to
p-nitrophenolate (pNP). A physiologically relevant co-
reagent (ascorbate or hydrogen peroxide) is required to
stimulate the redox chemistry of the Cu-ATCUN motif and
phenotype (Figure 1c). A shorter retention time (ca.
14.5 min) confirms the existence of this disaccharide, since
the amino column exhibits weaker affinity for disaccharides
than trisaccharides. Consistent with the results of pNP-fucose
cleavage, both CuGGH-tOL-NH2 and CuGGH-tOL-OH,
each with a shorter distance between the fucose-binding
domain and copper center, exhibit more robust cleavage
reactivity towards H-trisaccharide relative to the longer
CuGGH-OL-NH2 and CuGGH-OL-OH analogues (Fig-
ure 1d). CuGGH also displays cleavage reactivity, likely as
a result of nonselective cleavage, but it is significantly less
reactive than the metallopeptides containing a fucose-binding
domain.
H2 antigen on the cell surface was quantitated by using
a FITC-labelled antibody (Figure 2a). In the presence of
ascorbate, each of the artificial fucosidases significantly
reduced the FITC intensity, thus indicating removal of H2
antigen from the erythrocytes. We propose that copper redox
chemistry mediated by the metallopeptides promotes selec-
tive fucose cleavage from the H2 antigens and induces
regression of the H2 antigen to the precursor saccharides
corresponding to the Bombay blood type. The H2 antigens
remain intact in the absence of
[16]
promote the formation of metal-bound oxygen species. The
latter abstract hydrogen from the carbohydrate ring, which
[
17]
eventually leads to glycosidic bond cleavage.
All of the
metallopeptides with a fucose-targeting domain exhibit
a strong preference for l-fucose cleavage over d-glucose
(
Table 2 and Figure S5), which is abundant in blood and is
[
18]
commonly used for blood preservation. The lower KM for
the fucose substrate relative to glucose demonstrates selec-
tivity arising from the fucose-binding domain. The larger kcat
for the metallopeptides with truncated odorranalectin
CuGGH-tOL-NH and CuGGH-tOL-OH can be ascribed
2
to the shorter distance between the copper center and the
fucose-binding site relative to the analogues CuGGH-OL-
NH and CuGGH-OL-OH.
2
CuGGH, which lacks the fucose-targeting domain, exhib-
its no discrimination between l-fucose and d-glucose. These
results demonstrate that the targeting domain is a prerequisite
Table 2: Michaelis–Menten parameters for carbohydrate cleavage.
either copper or ascorbate, thus
[
a]
[a]
confirming that copper redox
chemistry is the origin of antigen
removal (Figure 2b). All of the
artificial fucosidases with a requisite
targeting domain were observed to
effectively remove H2 antigen, with
EC50 values of approximately 8–
Peptide sequence
pNP-a-l-fucoside
pNP-b-d-glucoside
À1
KM [mm]
kcat [min
]
kcat/KM
[
KM [mm]
kcat
[min
kcat/KM
[m min
À1
À1
À1
À1
À1
m
min
]
]
]
CuGGH-tOL-NH2 0.36Æ0.08 1.20Æ0.17 3360Æ310
CuGGH-tOL-OH 0.51Æ0.07 0.84Æ0.03 1680Æ240
CuGGH-OL-NH2 0.35Æ0.02 0.56Æ0.08 1630Æ310
2.25Æ0.62 0.51Æ0.08 245Æ34
2.94Æ0.31 0.93Æ0.28 376Æ25
2.16Æ0.08 1.07Æ0.13 514Æ37
2.12Æ0.17 0.32Æ0.08 150Æ31
CuGGH-OL-OH
CuGGH
0.42Æ0.06 0.44Æ0.05 1380Æ300
1.17Æ0.13 1.49Æ0.12 1270Æ36
0.72Æ0.18 1.40Æ0.34 1930Æ120
1
1 mm (Figure 2c,d and Figure S6).
The metallopeptides with an ami-
dated C terminus display improved
[
a] Reactions were performed with 5 mm metallopeptides, 1 mm ascorbate, and 1 mm H O in 50 mm
2 2
sodium phosphate buffer (pH 7.4) at 378C.
2
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Figure 2. Removal of the H2 antigen from erythrocytes. a) Flow cytom-
etry analysis of H2 antigen on the surface of human erythrocytes (red
blood cells, RBCs). After the erythrocytes were incubated with 20 mm
CuGGH-tOL-NH and 200 mm ascorbate for 4 h, immunofluorescent
2
staining was performed with a FITC-labelled anti-H2 antibody. b) Rela-
tive amount of H2 antigen on erythrocytes after incubation with or
without peptide or ascorbate. Solutions containing 200 mm ascorbate
and 20 mm CuGGH-tOL-NH were used. c) Relative amounts of H2
2
antigen on erythrocytes after incubation with various amount of
CuGGH-tOL-NH . d) The half-maximal effective concentration (EC )
2
50
for H2-antigen removal by artificial fucosidases in the presence of
Figure 1. Cleavage of the H2 antigen trisaccharide (H-trisaccharide), as
monitored by LC-ESI-MS. a) Removal of fucose from H-trisaccharide
200 mm ascorbate. e,f) Inhibitory effect of saccharides. Human eryth-
rocytes were incubated with 20 mm CuGGH-tOL-NH (e), or CuGGH
2
(
Fuca1-2Galb1-4GlcNAc) forms the disaccharide Galb1-4GlcNAc.
(f), with the indicated concentration of saccharides and 200 mm
b,c) A 25 mm solution of H-trisaccharide was incubated with 50 mm
CuGGH-tOL-NH , 1 mm ascorbate, and 1 mm H O at 378C for 0, 15,
ascorbate.
2
2
2
3
0, 60, 90, and 120 min. Extracted ion chromatogram (EIC) are shown
for m/z=530.2Æ0.1 (b) and m/z=384.1Æ0.1 (c). d) Time-dependent
cleavage of 25 mm H-trisaccharide by 50 mm metallopeptides, 1 mm
ascorbate, and 1 mm H O at 378C.
fucose and d-glucose was observed, which confirms that
removal of the H2 antigen by CuGGH arises from non-
selective cleavage (Figure 2 f).
2
2
In conclusion, we have established a convenient strategy
to design relatively short metallopeptides (16 to 20 amino
acids) as chimeric fucosidases. All of the metallopeptides we
designed exhibit selective carbohydrate cleavage of l-fucose
over d-glucose. Fucose cleavage from human blood H2
antigen trisaccharides results in regression to a disaccharide
product corresponding to the antigen of the Bombay blood
type. We expect that this strategy could be adopted to develop
glycosidases against other saccharide substrates of interest,
and that the artificial glycosidases developed with our
strategy could offer diverse applications complimentary to
conventional protein-based glycosidases. Fucose cleavage of
blood H2 antigen by artificial fucosidases leads to deletion of
the H2 antigen from the erythrocyte surface. The treated
erythrocytes could then potentially be applied as a substitute
for the rare Bombay blood type, and so our results represent
a first step to potentially addressing this problem.
reactivity toward H2 antigen, with an enhancement factor
around 18–31%, relative to analogues with a C-terminal
carboxylate. Amidation of the C terminus may reduce
repulsive interactions between the peptides and the nega-
tively-charged surface of the erythrocytes, however, the role
of the YASP residues remains unclear, because removal of the
YASP residues did not improve reactivity against H2 antigen.
In fact, the microenvironment on the erythrocyte surface and
interactions between the YASP residues and glycoproteins/
glycolipids may also influence the reactivity of the designed
metallopeptides. Removal of the H2 antigen by CuGGH was
also observed, although with lower efficiency (EC ꢀ 25 mm)
5
0
as a result of the lack of a targeting domain (Figure 2d and
Figure S6). Accordingly, the selective fucose cleavage reac-
tivity of our artificial fucosidases is most likely the origin of
H2 antigen removal. The addition of l-fucose inhibits the
binding of artificial fucosidases to cell-surface H2 antigen,
thereby suppressing cleavage (Figure 2e). The addition of d- Acknowledgements
glucose also impairs the fucosidase activity through non-
selective binding, but weaker inhibitory effects were observed
relative to l-fucose. When saccharides were applied to
CuGGH, no difference between the inhibitory effects of l-
This work was supported by a grant from the National
Institutes of Health [HL093446]. Z.Y. was supported by the
Ohio State University Pelotonia Fellowship Program.
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Conflict of interest
[
[
The authors declare no conflict of interest.
Keywords: blood antigens · bioinorganic chemistry ·
carbohydrates · enzymes · peptides
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Manuscript received: December 12, 2016
Final Article published: && &&, &&&&
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Communications
Artificial Enzymes
Z. Yu, J. A. Cowan*
&&& — &&&
Design of Artificial Glycosidases:
Metallopeptides that Remove H Antigen
from Human Erythrocytes
There will be blood: Metallopeptides were
designed as artificial fucosidases that can
induce selective cleavage of l-fucose over
d-glucose. Selective cleavage of fucose
(Fuca1-2Galb1-4GlcNAcb) enables
removal of the H2 antigen from human
erythrocytes, which could potentially
convert type-O blood into a substitute for
the rare Bombay blood type.
(
Fuc) from the H2 antigen trisaccharide
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ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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