A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases

Article abstract of DOI:10.1002/anie.202003105

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.

Full text of DOI:10.1002/anie.202003105

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: A Glycan Array-Based Assay for the Identification and
Characterization of Plant Glycosyltransferases
Authors: Colin Ruprecht, Max P. Bartetzko, Deborah Senf, Anna
Lakhina, Peter J. Smith, Maria J. Soto, Hyunil Oh, Jeong-
Yeh Yang, Digantkumar Chapla, Daniel Varon Silva, Mads H.
Clausen, Michael G. Hahn, Kelley W. Moremen, Breeanna R.
Urbanowicz, and Fabian Pfrengle
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202003105
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10.1002/anie.202003105
Angewandte Chemie International Edition
RESEARCH ARTICLE
A Glycan Array-Based Assay for the Identification and
Characterization of Plant Glycosyltransferases
Colin Ruprecht,[a],[c] Max P. Bartetzko,[a],[b] Deborah Senf,[a],[b] Anna Lakhina,[d] Peter J. Smith,[d] Maria J.
Soto,[d],[e] Hyunil Oh,[a],[b] Jeong-Yeh Yang, [d] Digantkumar Chapla,[d] Daniel Varon Silva,[a] Mads H.
Clausen,[f] Michael G. Hahn,[d] Kelley W. Moremen,[d] Breeanna R. Urbanowicz,*[d] and Fabian
Pfrengle*[a],[b],[c]
In memory of Professor Rolf Huisgen
[a]
[b]
[c]
Dr. C. Ruprecht, Dr. M. P. Bartetzko, Dr. D. Senf, H. Oh, Dr. D. Varon Silva, Prof. Dr. F. Pfrengle
Department of Biomolecular Systems
Max Planck Institute of Colloids and Interfaces
Am Mühlenberg 1, 14476 Potsdam, Germany
Dr. M. P. Bartetzko, Dr. D. Senf, H. Oh, Prof. Dr. F. Pfrengle
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Arnimallee 22, 14195 Berlin, Germany
Dr. C. Ruprecht, Prof. Dr. F. Pfrengle
Present address:
Department of Chemistry
University of Natural Resources and Life Sciences Vienna
Muthgasse 18, 1190 Vienna, Austria
E-mail: fabian.pfrengle@boku.ac.at
[d]
A. Lakhina, P. J. Smith, Dr. M. J. Soto, J.-Y. Yang, D. Chapla, Prof. Dr. K. W. Moremen, Prof. Dr. M. G. Hahn, Prof. Dr. B. R. Urbanowicz
Complex Carbohydrate Research Center
University of Georgia
315 Riverbend Road, Athens, GA 30602, USA
E-mail: breeanna@uga.edu
[e]
[f]
Dr. M. J. Soto
Present address:
US Department of Energy Joint Genome Institute (JGI)
Berkeley, CA, 94702, USA
Prof. Dr. M. H. Clausen
Center for Nanomedicine and Theranostics, Department of Chemistry
Technical University of Denmark
Kemitorvet 207, 2800 Kgs. Lyngby, Denmark
Supporting information for this article is given via a link at the end of the document.
Abstract: Growing plants with modified cell wall compositions is a
promising strategy to improve resistance to pathogens, increase
biomass digestibility, and tune other important properties. In order to
alter biomass architecture, a detailed knowledge of cell wall structure
and biosynthesis is a prerequisite. We report here a glycan array-
based assay for the high-throughput identification and
characterization of plant cell wall biosynthetic glycosyltransferases
(GTs). We demonstrate that different heterologously expressed
galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-
functionalized sugar nucleotide donors to selected synthetic plant cell
wall oligosaccharides on the array and that the transferred
monosaccharides can be visualized “on chip” by a 1,3-dipolar
cycloaddition reaction with an alkynyl-modified dye. The opportunity
to simultaneously screen thousands of combinations of putative GTs,
nucleotide sugar donors, and oligosaccharide acceptors will
dramatically accelerate plant cell wall biosynthesis research.
Introduction
As the global population increases, the demands for food, energy,
and materials will continue to grow dramatically, resulting in a
clear need for increased crop productivity and improved utilization
of biomass-derived bioenergy and sustainable bio-based
products and materials. Plant biomass, which is comprised of
carbohydrate-rich plant cell walls, represents the most abundant
renewable resource on Earth. Despite their ubiquity, major gaps
in our knowledge on the structure, function, and synthesis of the
cognate building blocks of plant cell walls remain. While the last
few years have seen significant advances in our understanding of
how plant cell walls are both constructed and decomposed, we
are still far away from tailoring plant cell wall composition and
architecture to our needs.[1] To identify potentially beneficial traits
that could be introduced into modern breeding varieties as well as
enhance the economic viability of lignocellulosic biomass as a
renewable resource, a detailed understanding of plant cell wall
architecture and biosynthetic pathways that are involved in its
construction are required.
1
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10.1002/anie.202003105
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RESEARCH ARTICLE
The main components of plant cell walls include a variety of
glycans, proteins, and phenolic polymers.[2] In these cell walls,
cellulose microfibrils are cross-linked by a group of highly complex
and heterogeneous polysaccharides, the hemicelluloses and
pectins. In plants, synthesis of cellulose occurs at the plasma
membrane, while the reminder of cell wall glycans are made in
the Golgi through glycosyltransferase (GT)-catalyzed additions of
monosaccharide residues from an activated nucleotide sugar
donor onto an acceptor, typically a saccharide, protein, or small
molecule.[3] The genome of the model plant Arabidopsis thaliana
encodes more than 561 GTs (nearly 2% of total genes) distributed
across 42 sequence-based families, identified thus far, in the
Carbohydrate-Active enZYme (CAZy) database,[3] and only a
handful have been biochemically validated. For example, despite
their enormous importance for cell wall biosynthesis and
structural properties, to date only 22 of the more than 100 GT
activities theoretically required for plant cell wall glycan synthesis
across all species have been confirmed via in vitro assays, largely
due to the historic difficulties associated with biochemical
characterization of enzymes involved in glycan synthesis.[4]
The ability of a putative GT to transfer a certain sugar
nucleotide to an acceptor substrate is most commonly evaluated
using MS,[5] HPLC,[6] or less accurate radioactivity-based
approaches.[7] However, as every reaction has to be performed
and analyzed individually, screening the overwhelmingly large
number of possible combinations of GTs, donor substrates, and
acceptor substrates becomes very difficult and impractical.
Glycan microarrays have become immensely powerful tools
for the high-throughput analysis of carbohydrate-protein
interactions,[8] but have not been widely applied for screening
carbohydrate-active enzymes such as GTs. Determining the
substrate specificities of GTs on glycan arrays is challenging, as
enzymes do not permanently bind to the immobilized acceptor
substrates and cannot directly be detected on the array. One
option is to use chemically functionalized sugar nucleotide donors
that enable a direct detection of the acceptor after transfer of the
modified glycosyl residue without the need for radiolabeled
donors.[9] This format allows for the use of a standard glycan array
platform with maximum throughput and sensitivity, suitable for
many different applications. It remains unclear if such unnatural
donor substrates will be accepted by all classes of GTs. However,
small modifications of sugar nucleotide donors including alkynyl-
and azido-modifications are usually tolerated well by GTs, as
observed in numerous metabolic glycan engineering studies, not
only in mammals and bacteria,[10] but also in plants.[11]
dye allows the simultaneous screening of thousands of individual
combinations of enzyme, donor, and acceptor (Scheme 1).
Scheme 1. Glycan array-based assay for the identification and characterization
of plant GTs. The array is incubated with a chemically modified nucleotide sugar
donor and
a putative GT, followed by visualization of any transferred
monosaccharide by an “on chip” reaction with an alkynyl-functionalized-dye.
Results and Discussion
Plant cell wall biosynthetic GTs are primarily transmembrane
proteins that for research purposes are commonly expressed in
eukaryotic systems such as yeast, tobacco leaves, or mammalian
cell lines.[4] The ability of these eukaryotic expression systems to
perform post-translational modifications is often required for
successful production and sufficient yield of active enzymes. A
particularly powerful method is the expression of putative GTs in
a soluble form (truncated to remove their transmembrane domain
and with an NH2-terminal secretion signal) in eukaryotic HEK293
cells.[18] HEK293 cell cultures have been proven to be a highly
successful system for robust expression of functional plant
glycosyl- and O-acetyltransferases.[5, 19] All enzymes studied in
this work were produced in this expression system.
We prepared 6- and 4-azido-functionalized (“clickable”) uridine
diphosphate (UDP) galactose donors 1 and 2 and 4-azido-xylose
donor 3 by coupling the respective per-acetylated sugar 1-
phosphates with cycloSal-activated uridine monophosphate
(Figure 1), a strategy that had proven successful for the
preparation of a range of natural and non-natural sugar
nucleotides.[20] The required sugar 1-phosphates were prepared
following literature reports or in analogy.[21] UDP-Gal derivatives
1 and 2 were subsequently reduced by hydrogenolysis to afford
the corresponding amino-functionalized derivatives 5 and 6 in
moderate purity (see supporting information).
We have recently developed a glycan array equipped with
88 synthetic plant oligosaccharides to determine the binding
epitopes of cell wall glycan-directed antibodies.[12] These
oligosaccharides represent fragments of natural hemicellulose,
hydroxyproline-rich glycoproteins, andpectic polysaccharides,
including arabinoxylan-,[13] type I and type II arabinogalactan-,[14]
xyloglucan-,[15] and mixed-linkage glucan-[16] related structures.[17]
This array is being continuously expanded with newly synthesized
structures to increase the covered chemical space. In
combination with chemically modified nucleotide donors, the
synthetic plant glycan array provides a powerful platform for
To test the ability of heterologously expressed plant GTs to
transfer functionalized UDP sugar derivatives to acceptor
substrates on our synthetic plant glycan array, the array was
incubated with UDP-N3-Gal derivatives 1 or 2 and the well-
characterized plant GT GALS1 from the model plant Arabidopsis
thaliana that transfers UDP-Gal to growing -1,4-galactan
developing
a
high-throughput screening method for the
identification and characterization of new plant GTs. Here we
report that incubation of this array with putative GTs and azido- or
amino-functionalized nucleotide sugars followed by visualization
of transferred monosaccharides by reaction with a functionalized
2
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10.1002/anie.202003105
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RESEARCH ARTICLE
sidechains in the pectic polysaccharide rhamnogalacturonan I
(RG-I) (Figure 2).[22]
oligosaccharide (see Supplementary Figure 2). Note that the
arrays were scanned with different photomultiplier gains. The
overall efficiency of donor transfer can be estimated using the
azide-controls printed in the bottom right corner of the array.
Amino-functionalized UDP-Gal derivatives 5 and 6 were also
transferred to the same oligosaccharides on the array and were
visualized using an NHS-azide crosslinker before reaction with
the alkynyl-functionalized dye. However, transfer was diminished
compared to UDP-N3-Gal derivatives 1 and 2.
Next, we analyzed related enzymes, such as PtGALS1 from
Populus and the close AtGALS1 ortholog AtGALS2.[24] Both
enzymes transferred the UDP-N3-Gal-donors 1 and 2, and only
very slight differences were observed in the pattern of recognized
acceptors compared to AtGALS1 (Figure 3A). Surprising results
were obtained when the array was incubated with UDP-6-N3-Gal
1 and AtGal31A, which was previously implicated in the synthesis
of -1,6-linked galactans in arabinogalactan proteins (AGPs).[25]
We found this GT to galactosylate substituted and unsubstituted
-1,3-galactan oligosaccharides rather than purely -1,6-linked
galactan oligosaccharides, indicating -1,3-galactosylation rather
than -1,6-galactosylation activity. Similar to GALS1,
galactosylation was dependent on the individual substitution
pattern of the acceptor substrates. We have also assayed three
fucosyltransferases (AtFUT4, AtFUT6, and AtFUT7) from
Arabidopsis using GDP-6-N3-Fuc derivative 4 and observed
fucosylation of essentially all oligosaccharides containing
arabinofuranose residues -1,3-linked to galactose. AtFUT4 and
AtFUT6 have previously been reported to fucosylate arabinose in
AGPs based on preliminary enzyme assays and the analysis of
knockout mutants.[26] The biochemical function of AtFUT7 was
previously unknown, and these data indicate that it shares similar
acceptor substrate specificity with AtFUT4 and AtFUT6, indicating
it is likely a previously undiscovered member of the AGP
biosynthesis pathway.
When we tested the Arabidopsis thaliana AtXXT1, an α-1,6-
xylosyltransferase involved in adding xylose to the β-1,4-glucan
backbone in xyloglucan,[27] for its ability to transfer UDP-4-N3-Xyl
derivative 3 to suitable acceptor substrates on the array, we did
not observe any enzymatic activity. Apparently, AtXXT1 did not
accept the azido-modification in 3. Instead, AtXXT1 was able to
transfer UDP-6-N3-Gal derivative 1 to a number of unsubstituted
and xylose-substituted glucan oligosaccharides (Figure 3B, see
also Supplementary Figure 3). AtXXT1 was found to prefer
unsubstituted and lowly substituted glucans over highly
substituted acceptors. Interestingly, not only purely -1,4-linked,
but also mixed linkage glucans (-1,3--1,4-linked) were
recognized by AtXXT1 when reacted with UDP-Gal-N3 derivative
1, as long as longer stretches of -1,4-linked Glc residues were
present. Also, polymeric hydroxylethylcellulose and natural
mixed-linkage glucan served as acceptor glycans. We were
surprised to see that, while the xylosyltransferase AtXXT1 was
Figure 1. Sugar nucleotide donors used in this study and their syntheses.
Any transferred galactose was subsequently visualized “on-chip”
with an alkynyl functionalized dye in a copper-catalyzed 1,3-
dipolar cyloaddition.[23] As expected, the reaction occurred
exclusively with -1,4-linked galactan oligosaccharides.
Interestingly, the enzyme discriminated between different
substitution patterns on the oligosaccharide acceptors. We
observed that -arabinofuranosyl substitutions in the 3-position of
some galactose residues (acceptors 51–53) were accepted by the
enzyme, while -galactosyl and -arabinofuranosyl substitutions
in the 6-position were not (acceptors 77–81). Both UDP-N3-Gal
derivatives were accepted as donor substrates, but 1 was
transferred more extensively, probably because transfer of 2 is
chain terminating due to the presence of N3 at the 4-position, while
1 can be transferred multiple times to the same acceptor
unable
to
transfer
UDP-Xyl-N3
derivative
3,
the
galactosyltransferase AtGALS1 did transfer UDP-Xyl-N3
derivative 3, albeit to a much lesser extent than UDP-Gal-N3.
Thus, due to the natural donor and acceptor substrate promiscuity
of these GTs, we have generated unnatural xylogalactan
oligosaccharides and galacto-mixed linkage glucan (-1,3--1,4-
linked) oligo- and polysaccharides on the array.
3
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10.1002/anie.202003105
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RESEARCH ARTICLE
Figure 2. A) AtGALS1-catalyzed transfer of azido- (1 and 2) and amino-functionalized (5 and 6) UDP-Gal derivatives to selected oligosaccharides on the array. B)
Printing pattern for the synthetic plant glycan array. Azide controls were printed in the bottom right corner of the array in 10, 100, 200, and 400 M concentrations.
Subset of -1,4-linked galactan acceptor substrates is shown.
To evaluate the possibility of assaying also mammalian GTs using
this glycan array platform,[18] we added three peptides containing
mammalian-type N-glycans (P1–P3)[28] to the array (Figure 3C).
These peptides were purified from egg yolk and enzymatically
trimmed. We incubated the array with UDP-Gal-N3 1 and 2 and
GDP-Fuc-N3 4 and the different GTs. HsFUT1 is a galactoside -
1,2-fucosyltransferase that is involved in ABO blood-group
antigen synthesis, primarily on red blood cells. Surprisingly, this
mammalian GT glycosylated a number of plant oligosaccharide
acceptors to a similar extent as the mammalian N-glycan
substrate. Besides galactose terminated N-glycan P2, a large
number of galactose-containing plant oligosaccharides, including
galactan and xyloglucan structures, were recognized. On the
other hand, -1,3-fucosyltransferases HsFUT6 and HsFUT7,
which are responsible for the synthesis of sialyl Lewis X
oligosaccharides in human cells, fucosylated non-mammalian
glycans only to a limited extent or not at all. While HsFUT6
recognized galactose terminated N-glycan P2, HsFUT7
fucosylated both P2 and de-galactosylated N-glycan P3. In
addition, HsFUT6 accepted chitin 104, galactosylated xyloglucan
36, and mixed-linkage glucans 38 and 41. None of the tested
mammalian fucosyltransferases accepted -2,6-sialylated glycan
P1. Human galactosyltransferase B4GALT5, which normally
extends Glc-ceramide to form lactosylceramide in animal cells,
galactosylated N-acetylglucosamine-terminated N-glycan P3 and
recognized chitin oligosaccharide 104 to a lesser extent. The
observed tolerance of some GTs for non-natural substrates may
be explored to enzymatically modify natural polysaccharides such
as xyloglucan, mixed-linkage glucan, and chitin to produce new
unnatural classes of polysaccharides with tailor-made physico-
chemical properties suited for applications in the materials
sciences.
4
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RESEARCH ARTICLE
Figure 3. A) Glycan array-based characterization of plant GTs PtGALS1, AtGALS2, AtGalT31A, and fucosyltransferases AtFUT4, AtFUT6, and AtFUT7. B) Glycan
array-based characterization of xylosyltransferase AtXXT1. AtXXT1 and AtGALS1 show loose donor substrate specificity and accept functionalized UDP-Gal and
UDP-Xyl donors, respectively. C). Selected human galactosyl- and fucosyltransferases recognize not only their natural acceptor substrates, but also fungal and
plant cell wall oligosaccharides. Subset of acceptor glycans is shown. The full set of printed glycans is presented in supplementary figure 1 (see supporting
information)
improved properties, including crop resistance to pathogens,
biomass digestibility, material strength, and the shelf life of fruits
Conclusion
and vegetables.
In conclusion, we have established a high-throughput assay for
the identification and characterization of plant cell wall
biosynthetic GTs based on the use of functionalized sugar
nucleotide donors on glycan microarrays that are equipped with
synthetic cell wall oligosaccharides. Utilizing glycan microarray
technology, the interactions of more than 100 different acceptor
oligosaccharides with several enzymes and sugar nucleotide
donors were investigated simultaneously on a single glass slide.
Current efforts are directed at the synthesis of further donor and
acceptor substrates to enable comprehensive screens for new
plant GT activities. Advances in plant cell wall biosynthesis
research will set the stage for production of tailor-made plants with
Acknowledgements
We gratefully acknowledge support from the Max Planck Society
and the German Research Foundation (DFG, Emmy Noether
program PF850/1-1 to F.P.). This work has also been supported
by the Center for Bioenergy Innovation (Oak Ridge National
Laboratory), a US Department of Energy (DOE) Bioenergy
Research Center supported by the Office of Biological and
Environmental Research in the DOE Office of Science and NIH
5
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RESEARCH ARTICLE
F. Andersen, I. Boos, C. Kinnaert, S. I. Awan, H. L. Pedersen, S. K.
Kracun, G. Lanz, M. G. Rydahl, L. Kjaerulff, M. Hakansson, R. Kimbung,
D. T. Logan, C. H. Gotfredsen, W. G. T. Willats, M. H. Clausen, Org.
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2017, 82, 12066-12084.
grants P41GM103390, R01GM130915 and P01GM107012.
M.H.C. is grateful to the Villum Foundation for support of the
PLANET Project (grant no. 9283). H.O. and D.V.S. thank the ERC
ETN Marie-Curie project GlyCoCan for financial support. We
thank Dr. Martina Delbianco for providing oligosaccharide 104.
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Keywords: • Carbohydrates • Plant Cell Wall • Glycan Array •
Glycosyltransferases • Sugar Nucleotides
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This article is protected by copyright. All rights reserved.

10.1002/anie.202003105
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Express and Screen. A high-throughput assay based on the application of azido-functionalized sugar nucleotide donors on a
synthetic glycan array for screening heterologously expressed plant glycosyltransferases was developed. The opportunity to express
and screen large numbers of glycosyltransferases instead of rationally selected candidates will markedly accelerate the elucidation of
plant cell wall biosynthetic pathways.
Twitter: @pfrenglelab
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This article is protected by copyright. All rights reserved.