Modified GM3 gangliosides produced by metabolic oligosaccharide engineering

Article abstract of DOI:10.1016/j.bmcl.2011.04.128

Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.

Full text of DOI:10.1016/j.bmcl.2011.04.128

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5006–5010
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modified GM3 gangliosides produced by metabolic oligosaccharide engineering
Chad M. Whitman a,b, Fan Yang a, Jennifer J. Kohler a,
⇑
a Division of Translational Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas,
TX 75390-9185, United States
b Department of Chemistry, Stanford University, Stanford, CA 94305-5080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 May 2011
Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialo-
sides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are
metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we
employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities
to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside produc-
tion, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the
two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data
suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and
excluded from others. Taken together, our results demonstrate that global analysis of sialoside produc-
tion can obscure sialoside-specific differences. These findings have implications for downstream applica-
tions of metabolic oligosaccharide engineering, including imaging and proteomics.
Keywords:
Ganglioside
Sialic acid
Diazirine
Sialyltransferase
Metabolic engineering
Ó 2011 Elsevier Ltd. All rights reserved.
Glycoconjugates are displayed on the surface of mammalian
cells where they play critical roles in both normal and pathological
recognition events. Efforts to better delineate glycoconjugates’
functions are hindered by the fact that the glycan components
are not primary gene products, limiting the utility of site-directed
mutagenesis to alter glycan structures in predictable ways. Meta-
bolic oligosaccharide engineering1,2 has provided an important
alternative approach to studying glycoconjugate function. By intro-
ducing small structural modifications to monosaccharides, and
then adding these analogs to cultured cells, it is possible to alter
the structure of cellular glycoconjugates in a predictable and con-
trolled way. Metabolic oligosaccharide engineering relies on the
abilities of mammalian cells to internalize analogs, metabolize
them, and incorporate the unnatural metabolites into glycan struc-
tures in place of natural sugars. Early work demonstrated that sia-
lic acid analogs in which the N-acyl chain was lengthened with
additional methylene units could be readily tolerated and incorpo-
rated into cell surface glycan structures in cells and living ani-
mals.3,4 Since then, metabolic oligosaccharide engineering has
expanded in scope and now enables the introduction of a variety
of functional groups with specialized reactivity (e.g., ketone,5
azide,6,7 alkyne,8 diazirine9) into various glycoconjugates, but most
successfully into sialic acid-containing glycoconjugates, also
known as sialosides.1 Metabolic oligosaccharide engineering has
found widespread utility, including the exploration of host–virus
interactions,10 the regulation of neuronal cell differentiation,11
and the visualization of developmental changes in glycosylation.12
While metabolic oligosaccharide engineering has proved to be a
highly effective method to alter sialoside structure, the majority of
studies measure the success of the engineering at a global level and
do not distinguish among the types of sialosides that are modified
by this technique. In humans, production of sialosides is controlled
by twenty different sialyltransferases.13 Sialyltransferases transfer
sialic acid from CMP-sialic acid, the nucleotide sugar donor, to
acceptor glycoconjugates. Sialyltransferases are classified into fam-
ilies that catalyze the formation of the
a2–3-, a2–6-, or a2–8-
linked sialic acid and each sialyltransferase has unique acceptor
specificity. Because of this diversity at the sialyltransfer step, it is
possible that metabolically incorporated sialic acid variants are
not equally well incorporated into all types of sialosides. Therefore,
we decided investigate the linkage-specific and acceptor-specific
incorporation of sialic acid analogs.
For these experiments, we employed analogs of N-acetylman-
nosamine (ManNAc), the biosynthetic precursor of sialic acid. In
mammalian cells, ManNAc is metabolized to N-acetylneuraminic
acid (NeuAc), a common form of sialic acid, in three enzymatic steps
(Scheme 1a). Similarly, N-acyl modified ManNAc analogs can be
metabolized to their sialic acid counterparts.1 Sialic acids and sialic
acid analogs are activated to CMP-sialic acids, then transported into
the Golgi, where the sialic acids are transferred to glycoconjugates
destined for secretion or cell surface display. Based on previous re-
ports of successful metabolic oligosaccharide engineering, we pre-
⇑
Corresponding author. Tel.: +1 214 648 1214; fax: +1 214 648 4156.
E-mail address: jennifer.kohler@utsouthwestern.edu (J.J. Kohler).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.128

C. M. Whitman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5006–5010
5007
OH
a
O
O
HO
HO
NH
2-O3PO
HN
O
UDP-GlcNAc
2-epimerase
O
O
P
O-
O
P
O-
-
HO
HN
OH
NeuAc-9-
2-O3PO
ManNAc-
6-kinase
CO2
O
R
HN
N
O
O
O
O
P-synthase
R
HO
HN
O
O
O
O
OH
HO
HO
HO
R
X
HO
HO
OH
OH
O
HO
UDP-GlcNAc
OH
ManNAc or analog
ManNAc-6-P or analog
NeuAc-9-P or analog
NeuAc-9-
P-phosphatase
HO
-
OH
HO
HO
sialyltransferase
CMP-NeuAc
CO2
-
OH
OH
O-CMP
CO2
CO2
(20 in humans)
synthase
HO
HN
O
HO
HN
HO
HN
O
O
-
O
R
OH
HO
glyco-
R
R
HO
HO
conjugate
O
O
O
glycoconjugate
sialylated proteins and lipids
(sialosides)
CMP-NeuAc or analog
NeuAc or analog
b
O
O
O
O
O
AcO
O
O
AcO
HN
AcO
HN
AcO
HN
AcO
HN
AcO
HN
AcO
OAc
CH3
Ph
N3
CH3
CH3
HN
HN
O
O
O
O
O
O
O
N
N
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
OAc
Ac4ManNAc
OAc
Ac5ManNGc
OAc
Ac4ManNProp
OAc
Ac4ManNBut
OAc
OAc
OAc
Ac4ManNPhAc
Ac4ManNAz
Ac4ManNDAz
Scheme 1. Metabolism of protected ManNAc analogs to sialosides. (a) BJAB K20 and CHO Lec3 cells lack UDP-GlcNAc 2-epimerase activity, rendering them unable to
biosynthesize ManNAc. When these cells are cultured in serum free conditions, they fail to produce sialosides. The UDP-GlcNAc 2-epimerase-deficient cells maintain the
ability to transform ManNAc to sialosides and can also metabolize exogenously supplied ManNAc analogs to their sialoside counterparts. (b) N-acyl-modified ManNAc
analogs were prepared and added to the media of cultured cells. All analogs were peracetylated to facilitate their entry into cells. All acetyl groups, including the one on the
N-glycolyl side chain of Ac5ManNGc, are believed to be removed by intracellular esterases.
pared a small panel of ManNAc analogs (Scheme 1b). ManNAz, a
widely-used azide-containing analog, is known to be metabolized
into SiaNAz, which has been incorporated into many cell surface
glycoconjugates,6 including gangliosides of Jurkat cells.14
ManNProp and ManNPhAc have also been successfully metabolized
to SiaNProp and SiaNPhAc, respectively, and incorporated into cell
surface gangliosides in several cancer lines. These modified ganglio-
sides have been utilized as immunogenic targets for antibodies.15–18
ManNBut is incorporated into glycoconjugates as SiaNBut, but acts
as a chain terminator in polysialylation.19 ManNGc, which can be
metabolized into the naturally occurring sialic acid NeuGc, has been
used to metabolically produce hydroxylated cell surface ganglio-
sides in neuronal cells to investigate myelin–axon interactions.20
Finally, ManNDAz is metabolized to SiaDAz, which contains a
photoactivable crosslinking group that enables covalent crosslink-
ing of sialosides to their binding partners.9,21 In the experiments de-
scribed here, all ManNAc analogs were peracetylated to enable their
facile transport into the cell.22 Protecting groups are assumed to be
removed by intracellular nonspecific esterases.
To specifically track the metabolism of exogenously introduced
ManNAc analogs, we employed two cell lines, human BJAB K2023,24
and hamster CHO Lec3,25 that are impaired in sialic acid biosynthe-
sis due to lack of UDP-GlcNAc 2-epimerase activity (Scheme 1a).
When these epimerase-deficient cell lines are cultured in serum-
free conditions, where they cannot scavenge sialic acid, they are
unable to synthesize sialosides. When the media is supplemented
with Ac4ManNAc, the cells regain the ability to generate sialylated
glycoconjugates. Similarly, when the cells are cultured with unnat-
ural ManNAc analogs, any sialoside production is interpreted to re-
flect successful metabolism of these unnatural analogs to
sialosides. As a control, we also examined sialoside production in
similar cell lines, BJAB K88 and wt CHO, that maintain UDP-GlcNAc
2-epimerase activity.
ined total cell surface sialoside levels using the recently reported
periodate oxidation and aniline-catalyzed oxime ligation (PAL).26
Consistent with previous reports, we found that Ac4ManNAc,
Ac4ManNProp, and Ac4ManNBut were robustly metabolized to
sialosides, both in human BJAB K20 cells and hamster CHO Lec3 cells
(Fig. 1a and b). Ac4ManNAz and Ac4ManNDAz were also metabolized
to sialosides in both cell lines, albeit at lower efficiency. Our data do
not clearly demonstrate that Ac4ManNPhAc is metabolized to sialo-
sides, but this may reflect the sensitivity limits of the PAL assay since
SiaNPhAc-containing glycoconjugates have been detected using
antibodies by others.16–18 We noted one significant difference be-
tween human BJAB K20 cells and hamster CHO Lec3 cells cultured
with Ac5ManNGc: Lec3 cells produce high levels of NeuGc-contain-
ing glycoconjugates, while these levels are lower in K20 cells.
Next, we used lectin binding to examine whether the incorpora-
tion of variant sialic acids was linkage-dependent (Fig. 1c–e).
a
2–3-Linked sialosides were detected with the Maackia amurensis
lectin (MAA) and 2–6-linked sialosides were detected with the Sam-
bucus nigra lectin (SNA). In human BJAB K20 cells, we detected both
2–3- and 2–6-linked sialosides. Generally, the linkage-specific
a
a
a
incorporation trends were similar to the global incorporation trend
observed by PAL. However, while a relatively low level of MAA bind-
ing was observed in BJAB K20 cells cultured with Ac5ManNGc, the le-
vel of SNA binding was high and exceeded the level of SNA binding
observed for BJAB K20 cells cultured with Ac4ManNAc. In CHO Lec3
cells, weexaminedonly
cells do not produce detectable levels of
a
2–3-linkedsialosideproduction, since these
a
2–6-linked sialosides.27 In
CHO Lec3 cells, the trends we observed for MAA binding for different
ManNAc analogs were similar to the trends for global sialoside pro-
duction detected by PAL. However, weonce again noticed a difference
between BJAB K20 and CHO Lec3 cells: our MAA binding data sug-
gested that CHO Lec3 cells produce high levels of NeuGc-containing
a2–3-linked sialosides, while these levels are lower in BJAB K20 cells.
BJAB K20 and CHO Lec3 cells were cultured in serum-free condi-
tions with each of the protected ManNAc analogs. First, we exam-
We considered the possibility that the observed differences SNA and
MAA binding that we observed for BJAB K20 cells cultured with

5008
C. M. Whitman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5006–5010
BJAB cells
CHO cells
a
b
K88
K20
wt
Lec3
4000
3000
2000
2500
2000
PAL analysis
(sialic acid)
1000
0
1000
0
c
e
d
wt
Lec3
K88
K20
60
40
20
0
80
60
40
20
0
MAA Binding
(α2-3-linked)
K88
K20
1200
800
400
0
SNA Binding
(α2-6-linked)
Figure 1. Cell surface display of modified sialic acids. Cell surface sialylation of engineered BJAB K20 and CHO Lec3 cells was analyzed by periodate oxidation and aniline-
catalyzed oxime ligation (PAL; measures total cell surface sialylation) and by binding of fluorescently-labeled lectins (MAA recognizes 2–3-linked sialic acid; SNA recognizes
2–6-linked sialic acid). Fluorescence of individual cells, reporting on cell surface sialylation, was measured by flow cytometry. The mean fluorescence intensity (MFI) of at
a
a
least 10,000 live cells is shown. BJAB K88 and wt CHO cell lines possessing UDP-GlcNAc 2-epimerase activity were used as positive controls. Experiments were performed in
biological triplicate; duplication of the entire experiment yielded similar results. (a) PAL analysis of BJAB cells. (b) PAL analysis of CHO cells. (c) MAA lectin analysis of BJAB
cells. (d) MAA lectin analysis of CHO cells. (e) SNA lectin analysis of BJAB cells. SNA binding was not assayed in CHO cells because they lack production of
sialosides.
a2–6-linked
Ac5ManNGc reflect an altered lectin affinity for NeuGc-containing
sialosides, but feel that this is unlikely. First, previous reports demon-
strated that SNA and MAA lectins do not discriminate between NeuAc
and NeuGc.28 Second, CHO Lec3 cells cultured with Ac5ManNGc pro-
duce high levels of MAA binding, suggesting that MAA is capable of
binding NeuGc-containing glycoconjugates with high affinity. There-
fore, we favor the interpretation that BJAB K20 cells produce NeuGc-
Lec3 cells in serum-free conditions with each of the protected Man-
NAc analogs. Metabolically-labeled cells were harvested and the
gangliosides were extracted using established methods.30–32
Gangliosides were resolved by high performance thin layer chroma-
tography (HPTLC) and visualized by resorcinol staining, which
specifically detects sialic acid-containing molecules.
BJAB K20 cells readily produced GM3 analogs containing a vari-
ety of modifications to the N-acyl side chain of sialic acid (Fig. 2a).
Ac4ManNAc, Ac4ManNProp, Ac4ManNBut, and Ac4ManNAz were
efficiently metabolized to the corresponding GM3 analogs. Ac4-
ManN DAz was also metabolized successfully, albeit less efficiently.
However, when BJAB K20 cells were cultured with Ac5ManNGc, pro-
duction of NeuGc-containing GM3 was significantly lower than pro-
duction of NeuAc-containing GM3. This result is consistent with the
MAA binding experiments that suggest that NeuGc is not efficiently
containing
do.
a2–3-linked sialosides less efficiently that CHO Lec3 cells
To further investigate the differences in
production in these two cell lines, we chose to examine production
of a single 2–3-linked sialoside produced by a single sialyltransfer-
a2–3-linked sialoside
a
ase. GM3 is a sialylated glycolipid, also known as a ganglioside,
produced when the sialyltransferase ST3GAL5 adds sialic acid in
an
a
2–3-linkage to lactose ceramide (LacCer) (Scheme 2).29 Both
BJAB K20 and CHO Lec3 cells express ST3GAL5 and, when provided
with sialic acid, are capable of producing GM3. Moreover, GM3 is
not elaborated to more complex gangliosides at significant levels
in either of these cell lines, simplifying the analysis of unnatural sia-
lic acid incorporation levels. Again, we cultured BJAB K20 and CHO
incorporated into the a2–3-linkage in this cell line. Only one analog
(Ac4ManNPhAc) was not metabolized to a GM3 analog at a level
detectable by resorcinol staining. MALDI-TOF-MS analysis (Supple-
mentary data) of extracted gangliosides from BJAB K20 cells cul-
tured with Ac4ManNAc, Ac5ManNGc, Ac4ManNAz, and Ac4ManN

C. M. Whitman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5006–5010
5009
O
DAz confirmed the identity of metabolically engineered GM3
molecules.
CH3
OH
OH
HN
HO
n
O
O
O
We observed a different pattern of GM3 analog production in
CHO Lec3 cells (Fig. 2b). CHO Lec3 cells readily metabolized
Ac4ManNAc, Ac5ManNGc, and Ac4ManNProp to the corresponding
GM3 analogs. Increasing the side chain length (Ac4ManNBut and
Ac4ManNAz) led to diminished levels of incorporation and larger
N-acyl side chains, found on Ac4ManNPhAc and Ac4ManNDAz,
were not tolerated. MALDI-TOF-MS analysis of extracted ganglio-
sides from CHO Lec3 cells cultured with Ac4ManNAc, Ac5ManNGc,
and Ac4ManNAz confirmed the identity of metabolically engi-
neered GM3 molecules (Supplementary data). In comparison to
BJAB K20 cells, CHO Lec3 cells appear to be more efficient at incor-
porating NeuGc into GM3 and less efficient at incorporating
SiaNBut and SiaNAz.
O
HO
HO
CH3
HO
12
OH
OH
OH
LacCer
OH
O-CMP
CO2
HO
HN
O
-
R
HO
O
CMP-NeuAc
analog
ST3GAL5
The experiments presented here allowed us to compare the glo-
bal versus specific sialoside production in two mammalian cell lines.
By using cell lines deficient in sialic acid biosynthesis, we specifically
tracked production of modified sialosides. Our data reveal sialoside-
specificincorporation patternsthat depend on the form of sialicacid.
For example, BJAB K20 cells seem to discriminate against incorpo-
CMP
O
CH3
n
HO
–OH
OH
CO2
HN
HO
OH
HO
HO
O
O
O
O
O
O
HO
CH3
12
HN
R
OH
OH
OH
rating NeuGc into
a2–3-linked sialosides, including GM3. The low
O
levels of NeuGc incorporation into
a2–3-linked sialosides are unli-
GM3 analog
kely to be explained solely by lower levels of CMP-NeuGc produc-
tion, since the BJAB K20 cells cultured with Ac5ManNGc still show
robust levels of SNA binding. Similarly, CHO Lec3 cells produce rela-
tively high levels of cell surface SiaNBut, but are ineffective at pro-
ducing SiaNBut-containing GM3. These differences between global
and specific sialoside production may reflect differences in the activ-
ity or expression level of one or more of the enzymes involved in
sialoside metabolism. Perhaps certain sialyltransferases discrimi-
nate among different CMP-sialic acid analogs, resulting in variant
sialic acids being more readily incorporated into some sialosides,
but excluded from others. Alternatively, discrimination may occur
at the level of sialoside catabolism, with sialidases exhibiting sub-
strate specificity for different forms of sialic acid. In addition, differ-
ing rates at which different sialosides are synthesized and degraded
may affect our observations.
Scheme 2. Biosynthesis of GM3 gangliosides. GM3 is produced by the action of a
single sialyltransferase, ST3GAL5, that transfers sialic acid from a CMP-sialic acid to
LacCer.
a
K88
K20
GM3
GM3
GM2
GM2
GM1
GM1
Our findings also highlight key differences in the utility of dif-
ferent cell lines for performing metabolic oligosaccharide engi-
neering experiments, particularly those aimed at producing
modified gangliosides. The BJAB K20 cell line demonstrates the
substrate flexibility to introduce larger modifications (such as the
N-butanoyl substituent and the diazirine) into gangliosides, but
shows attenuated ability to introduce a naturally occurring sialic
acid variant (NeuGc). Conversely, the CHO Lec3 cell line exhibits
restrictions in substrate scope but, unlike the BJAB cell line, is able
to utilize Ac5ManNGc as a precursor for NeuGc-GM3 production.
We do not know the molecular basis of the specificity differences
between the BJAB K20 and CHO Lec3; these cell lines differ in both
their tissue and species of origin. However, we are intrigued by
these findings in light of the fact that BJAB K20 cells are derived
from humans, an organism that produces NeuAc but not NeuGc,
while CHO Lec3 cells are derived from hamsters, an organism that
produces both NeuAc and NeuGc.33
wt
Lec3
b
GM3
GM3
GM2
GM1
GM2
GM1
Acknowledgments
Figure 2. HPTLC analysis of gangliosides produced by BJAB and CHO cells cultured with
protected ManNAc analogs. BJAB K20 and CHO Lec3 cells were cultured with
protected ManNAc analogs. BJAB K88 and wt CHO cell lines containing UDP-GlcNAc
2-epimerase activity were used as positive controls. Gangliosides were extracted,
resolved by HPTLC, and visualized by resorcinol staining. Mobilities of standard
gangliosides are indicated on either side of the HPTLC images. Gangliosides appear
as doublets, reflecting two different fatty acid lengths that are incorporated into
LacCer in these cell lines. (a) BJAB cells generate GM3 and small quantities of GM1a
gangliosides. (b) CHO cells produce only GM3 gangliosides. Gangliosides incorpo-
rating modified sialic acids have altered mobilities. Incorporation of the modified
sialic acids was confirmed by mass spectrometry (Supplementary data).
We thank Michael Pawlita (German Cancer Research Center) and
James Paulson (The Scripps Research Institute) for sharing BJAB K20
and BJAB K88 cells, Mark Lehrman (UT Southwestern Medical Cen-
ter) for sharing wt CHO cells, and Pamela Stanley (Albert Einstein
College of Medicine) for sharing CHO Lec3 cells. We thank Yan Li
(UT Southwestern Medical Center Protein Chemistry Technology
Center) for mass spectrometry analysis of sugars, Parastoo Azadi
and Roberto Sonon (University of Georgia Complex Carbohydrate

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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.128.
References and notes
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