On-virus construction of polyvalent glycan ligands for cell-surface receptors

Article abstract of DOI:10.1021/ja077801n

Glycans arrayed on the exterior of virus particles were used as substrates for glycosyltransferase reactions to build di- and trisaccharides from the virus surface. The resulting particles exhibited tight and specific associations with cognate receptors on beads and cells, in one example defeating in cis cell-surface interactions in a manner characteristic of polyvalent binding. Combined with the ability of viruses to provide structurally well-defined attachment points, the methodology provides a convenient and powerful way to prepare complex carbohydrate ligands for clustered receptors. Copyright

Full text of DOI:10.1021/ja077801n

Published on Web 03/15/2008
On-Virus Construction of Polyvalent Glycan Ligands for Cell-Surface
Receptors
Eiton Kaltgrad,† Mary K. O’Reilly,‡ Liang Liao,‡ Shoufa Han,‡ James C. Paulson,*,‡ and M. G. Finn*,†
Department of Chemistry and The Skaggs Institute for Chemical Biology, and Departments of Chemical Physiology
and Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received October 10, 2007; E-mail: mgfinn@scripps.edu
Cell surface receptors that recognize glycans as ligands mediate
a vast range of events in cellular biology through low-affinity,
multivalent (simultaneous multipoint) binding interactions.1 The
development of ligand-based probes of these receptors must there-
fore address both the challenges of glycan synthesis and the need
for multivalent platforms. A polyvalent display of glycan ligands
is usually achieved by synthesis of the desired glycan with a linker
containing a functional group at the reducing end, allowing conju-
gation to a polyvalent platform bearing the complementary reactive
group.2 An alternative approach of “immobilizing” glycan reaction
precursors to the platform of choice and “building out” from such
Figure 1. (Top) Structures of glycan azides used to make the “preformed”
virus conjugates. (Bottom) Preparation and use of alkyne-labeled CPMV
and Qâ.
surfaces to make polyvalent oligosaccharides would provide a way
to make complex and expensive oligosaccharides precisely placed,
as has recently been demonstrated using gold nanoparticles.3 Here
we report the implementation of this strategy for labeling the
exterior surface of a virus capsid, taking advantage of the com-
patibility of the protein particles with glycosyltransferase enzymes
and chemoenzymatic reaction conditions.4 The resulting structures
comprise multivalent arrays of glycan ligands favorable for binding
to a cell surface glycan receptor in a highly specific manner.
Our target to probe the viability of on-virus oligosaccharide
synthesis was CD22 (Siglec-2), a receptor specifically expressed
on B cells and B lymphoma cells that is involved in regulation of
B cell signaling. The natural ligand of CD22 is SiaR2-6Gal, which
is found at the termini of N-linked glycans on B and T cells.5 CD22
is bound by ligands presented by neighboring molecules on the
same B cell surface, an interaction “in cis” that promotes strong
binding even though the ligand-receptor pair has low intermo-
Figure 2. Sequential enzymatic synthesis of CPMV-BPCsial, monitored
lecular affinity (Kd ) ca. 0.1-0.2 mM).6 Binding of other cells to
by lectin recognition properties. CPMV-GlcNAc (top), CPMV-LacNAc
(center), or CPMV-BPCsial (bottom) were incubated with magnetic beads
bearing Erythrina cristagalli agglutinin (ECA, specifically binds terminal
galactose16) or a recombinant CD22-Fc immunoglobulin chimera (hCD22,
specifically binds BPCsial) and analyzed by flow cytometry.
the CD22 receptor is thereby inhibited unless the competing
sialosides are presented in an effective polyvalent fashion.
The 9-biphenyl carbonyl (BPC) derivative7 of the natural
sialoside ligand, SiaR2-6Galâ1-4GlcNAc, was selected as our target
ligand for construction of multivalent capsid-based structures.5
GlcNAc, the intermediate Galâ1,4-GlcNAc (LacNAc), and the final
BPC sialoside were prepared as O-linked azidoethyl derivatives 1,
2, and 3, respectively (Figure 1).4c,8 The conveniently accessible
azide groups were used to array the carbohydrates on alkyne-
decorated polyvalent particles by the CuI-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction.9 Two particles were employed:
the cowpea mosaic virus (CPMV)10 and bacteriophage Qâ coat
protein,11 both icosahedral structures approximately 30 nm in
diameter.12 Alkyne groups were introduced by acylation of surface
lysine side chains with alkynyl N-hydroxysuccinimide ester 4.13
Wild-type Qâ, having more subunits per particle (180) than CPMV
(60) and approximately the same number of surface-accessible
lysines per subunit, displays a greater number of reactive sites. To
make a particle in which the location of the attachment points is
precisely known, the Qâ sequence was modified by the replacement
of lysine at position 16 with methionine (K16M). This protein was
expressed in a methionine auxotroph of E. coli with addition of
the alkyne-containing unnatural amino acid homopropargyl glycine
(HPG), resulting in incorporation of HPG at approximately 90 of
the 180 newly introduced Met sites at position 16. This chimera,
designated K16HPG, is described more fully elsewhere.14
Compounds 1, 2, and 3 were arrayed on the virus scaffold by
azide-alkyne cycloaddition under the influence of precatalyst 6,
in a method previously shown to give complete coverage of the
displayed alkyne groups.9 The resulting glycan-derivatized particles
were designated V-GlcNAc, V-LacNAc, and V-BPCsial, respec-
tively, where “V” is either CPMV or Qâ. The number of attached
glycans in each case was estimated by analogy to reactions
performed under identical conditions using a selenium-containing
molecule that can be quantitatively detected after attachment.15
The alternative strategy of carrying out the synthesis directly on
the V-GlcNAc particle was implemented as shown in Figure 2.
Enzymatic condensation of CPMV-GlcNAc with uridine diphos-
pho-galactose (UDP-galactose, generated in situ by rapid galac-
toepimerase-catalyzed conversion of the inexpensive UDP-glucose)
† Department of Chemistry and The Skaggs Institute for Chemical Biology.
‡ Departments of Chemical Physiology and Molecular Biology.
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10.1021/ja077801n CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
GlcNAc was converted to CPMV-BPCsial in an efficient “one-
pot” reaction (Figure 2). The particles obtained after purification
from the cascade reaction were nearly as effective in binding to B
cells as the particles obtained by CuAAC conjugation of presyn-
thesized BPC-sialoside to the virus scaffold (Figure 3D).
The results shown here demonstrate that virus capsids provide a
platform that can be used to create a multivalent particle by en-
bloc transfer of preformed ligands or used as “beads” upon which
multivalent ligands can be synthesized by sequential enzymatic
transformations. Their unique size, density, and chemically robust
nature allow for convenient isolation away from other reaction
components. Most importantly, viruses allow the precise placement
of functional units, in this case the starting glycan acceptors, and
therefore control of the distribution of polyvalent oligosaccharides
obtained from such “on-bead” transformations. The resulting
particles can be highly effective binders to specific receptor clusters,
as in the present case to cell-surface CD22.
Figure 3. Flow cytometry analyses of the binding of CPMV-glycans with
native cells in a CD22- and sialic acid-dependent manner. (A and B) CPMV-
LacNAc (gray filled) or CPMV-BPCsial (black) incubated with CHO or
CHO-CD22 cells. (C) CPMV bearing LacNAc (gray filled) or BPCsial (thick
black solid) incubated with native Raji cells. BPCsial-bearing viruses were
also incubated in the presence of the indicated concentrations of free BPC-
sialoside 3 as inhibitor. (D) Raji cells incubated with wild-type CPMV (light
gray filled), CPMV-LacNAc (dashed), CPMV-BPCsial from preformed
trisaccharide (thick black solid), CPMV-BPCsial made enzymatically on
the virus from preformed CPMV-LacNAc (dotted), or CPMV-BPCsial
made in a one-pot reaction on the virus (thin black solid).
gave CPMV-LacNAc. The product was purified away from the
small molecule and enzymatic reagents by ultracentrifugation
through a 10-40% sucrose gradient, which is typical for virus
particles of this size and nearly as convenient as filtration of poly-
styrene resin beads. Similarly, reaction of CPMV-LacNAc with
BPC-sialic acid 7 gave the final product CPMV-BPCsial, which
was again purified by sucrose gradient sedimentation. The yield of
recovered virus for each step was approximately 70%, consistent
with the normal loss of material sustained in manipulations of this
kind.
Acknowledgment. We thank the Skaggs Institute for Chemical
Biology, the NIH (CA112075, AI056013, GM60938), the American
Cancer Society (PF0719301), the W. M. Keck Foundation for
support of this work, and Warren Wakarchuck at the National
Research Council, Ottawa for galactosyltransferase.
Supporting Information Available: Synthetic and analytical
procedures and data. This material is available free of charge via the
Internet at http://pubs.acs.org.
The viruses derived from conjugation of presynthesized and
sequentially formed glycans were found to be intact with no
indication of decomposition or instability.15 The binding properties
of the particles following enzymatic conversion were assessed by
flow cytometry using magnetic beads coated with glycan binding
proteins that recognize the precursor and product of each reaction
(Figure 2). Complete selective switching between the expected
binding properties was observed with each enzymatic transforma-
tion. Very similar results were obtained with the analogous Qâ
particles,15 showing that polyvalent glycan-lectin binding was
independent of the platform.
The virus conjugates were also found to bind to CD22 expressed
on cell surfaces. CPMV-BPCsial, but not CPMV-LacNAc,
displayed preferential binding to Chinese hamster ovary (CHO) cells
expressing recombinant CD22 (Figure 3B) and to Raji lymphoma
cells, which naturally express CD22 (Figure 3C), over native CHO
cells that do not express CD22 (Figure 3A). Competition for binding
to Raji cells between virus conjugates of BPCsial and free BPC-
sialoside 3 showed partial disruption of the virus-cell interaction
at 100 µM of the monomeric ligand, and greater, but still incomplete
inhibition at 1 mM (Figure 3C and Supporting Information). These
values are 4-40 times that of the total BPC-sialoside concentration
presented on V-BPCsial (ca. 25 µM) and far greater than the conf-
centration of glycosylated particles (ca. 130 nM), consistent with
the expectation that interaction of virus-displayed ligands with CD22
on cells is polyvalent in nature. Similar tight-binding behavior was
observed for BPC-sialoside adducts of wild-type and K16HPG
forms of Qâ, which differ in the number of attached glycans.15
Such a result suggests that the valency presented on these constructs
exceeds that needed to form a stable complex with CD22 on the
cell surface.
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