Glycosynthase-mediated synthesis of glycosphingolipids

Article abstract of DOI:10.1021/ja058469n

Glycosphingolipids play crucial roles in virtually every stage of the cell cycle, and their clinical administration has been proposed as a treatment for Alzheimer's, Parkinson's, stroke, and a range of other conditions. However, lack of supply has severely hindered testing of this potential. A novel glycosynthase-based synthetic strategy is demonstrated, involving a mutant of an endoglycoceramidase in which the catalytic nucleophile has been ablated. This mutant efficiently couples a range of glycosyl fluoride donors with a range of sphingosine-based acceptors in yields around 95%. This technology opens the door to large-scale production of glycosphingolipids and, thus, to clinical testing. Copyright

Full text of DOI:10.1021/ja058469n

Published on Web 04/20/2006
Glycosynthase-Mediated Synthesis of Glycosphingolipids
†
§
§
§
‡
Mark D. Vaughan, Karl Johnson, Shawn DeFrees, Xiaoping Tang, R. Antony J. Warren, and
,
†
Stephen G. Withers*
Protein Engineering Network of Centres of Excellence, Departments of Chemistry and Microbiology, UniVersity of
British Columbia, VancouVer, British Columbia, Canada, and Neose Technologies, Inc.,
Horsham, PennsylVania
Received December 13, 2005; E-mail: withers@chem.ubc.ca
Glycosphingolipids, particularly the sialic acid-containing gan-
gliosides, have numerous crucial functions within fundamental
processes of human physiology. Predominantly localized to neuronal
cell membranes (although present in all mammalian cell mem-
branes), these compounds are extremely important in biochemical
signaling and appear to play a critical role in virtually every stage
of the cell life cycle, including growth, proliferation, differentiation,
adhesion, senescence, and apoptosis.1 Recent evidence suggests
that clinical administration of particular gangliosides may alleviate
Scheme 1. Reactions Catalyzed by Wild-Type (a) and
Glycosynthase (b) EGC II Enzymes
-6
7
4
symptoms of cancer and diabetes and may also promote nerve
8
,9
tissue regeneration, which can counter the effects of Alzhei-
1
0,11
and Parkinson’s12 diseases, stroke, fetal alcohol syn-
13
mer’s
drome,14 and autoimmunity in transplant cases. As such, there is
great potential for health benefits stemming from the therapeutic
utilization of these glycolipids.
15
A major limiting factor in applying these glycolipids to disease
treatment is the great difficulty associated with producing large
quantities of pure samples in an efficient and economical fashion.
Commercial suppliers of gangliosides rely on that isolated from
natural sources such as bovine brain and canine blood, which is
impractical for large-scale preparation and also poses risks for
possible contamination by prions or other infectious agents.
Chemical syntheses of gangliosides have been reported;1
however, significant challenges exist in these approaches with
regard to control of stereo- and regiochemistry, the need for multiple
protection/deprotection steps, poor product yields, difficult purifica-
tion, and the unsuitability for industrial production.
Enzymatic syntheses offer significant improvements over purely
chemical routes in the preparation of glycosylated compounds due
to the ability to form specific glycoside linkages in the presence of
many chemically similar hydroxyl groups. The most obvious route
to the enzymatic synthesis of glycosphingolipids would be to
recreate the natural biosynthetic pathway, which involves the
sequential addition of glycosides to the core lipid acceptor.20 This
route, however, is problematic. First, the glucosyl transferase
responsible for transferring the first carbohydrate residue is not
currently available. Additionally, all subsequent transfer steps would
have to be performed on the hydrophobic glycolipid, which could
present problems in scale-up and product isolation.
lipid and sugar moieties (Scheme 1a).2
1-24
This enzyme appeared
6-19
to be a good candidate for conversion to a synthetic enzyme using
glycosynthase methodology.
The glycosynthase approach utilizes retaining glycosidase vari-
ants in which the catalytic nucleophilic residue has been replaced
by a residue unable to perform this function. When supplied with
glycosyl fluorides with anomeric configuration opposite that of the
normal cleaved linkage, these enzymes are often able to transfer
the glycosyl moiety to an appropriate acceptor.25 In this study, the
parent glycosidase from which the glycosynthase was generated
was endoglycoceramidase II (EGC II) from Rhodococcus strain
M-777.2
1-24
The Escherichia coli codon-optimized gene for this
enzyme (minus 30 N-terminal residues constituting a secretion
signal sequence) was synthesized (Blue Heron Biotechnology, Inc.)
and expressed at high levels (180-200 mg protein per liter of
culture) in an Escherichia coli host.
On the basis of sequence alignments, EGC II has been assigned
to glycosyl hydrolase family 5, which predominantly consists of
cellulases (â-1,4-endoglucanases). Previous studies in this laboratory
have permitted the identification of the catalytic nucleophile residue
in this family.26 Sequence similarities in the region of the nucleo-
phile allowed us to tentatively assign this function to Glu351 in
EGC II. Substitution of this residue with a series of residues
expected to be catalytically incompetent in a hydrolytic context
We envisioned an alternative approach to the synthesis, involving
preassembly of the glycosyl headgroup using known glycosyl
transferases under normal aqueous conditions, followed by transfer
of the oligosaccharide to the lipid acceptor. Although there are no
known glycosyl transferases that catalyze this reaction, there is a
well-studied endoglycoceramidase enzyme that catalyzes the reverse
reaction, i.e. the hydrolysis of glycosphingolipids to their constituent
(
Ser, Ala, Gly, and Asp) led to the loss of EGC II activity,
supporting the postulated role for this residue in the mechanism.
When 3′-sialyllactosyl fluoride (1) and D-erythro-sphingosine (2)
were combined at 5 mM each in 25 mM sodium acetate, pH 5.0
containing 0.2% (v/v) Triton X-100 and incubated in the presence
†
Protein Engineering Network of Centres of Excellence, Department of
Chemistry, University of British Columbia.
‡
Protein Engineering Network of Centres of Excellence, Department of
Microbiology.
§
Neose Technologies, Inc.
6300
9
J. AM. CHEM. SOC. 2006, 128, 6300-6301
10.1021/ja058469n CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Reaction Yields for EGC II E351S Glycosynthase
by acylation of the lyso analogue using N-palmitoyl succinimide
as an acyl donor.
Reactions with Various Glycosyl Fluoride Donor Sugarsa
The ability of this enzyme to form the synthetically challenging
glycosidic bond between the oligosaccharide and lipid portions of
gangliosides and related glycolipids offers a new route to the
efficient assembly of glycosphingolipids, which should provide
avenues for the large-scale synthesis of these therapeutically
valuable compounds.
Acknowledgment. We thank Emily Kwan for technical as-
sistance, Dr. Hongming Chen for synthesis of substrates, Dr.
Krisztina Paal for advice, Dr. Warren Wakarchuk (NRC Canada
Institute for Biological Sciences) for supplying glycosyl transferase
enzymes, and the Protein Engineering Network of Centres of
Excellence (PENCE) for financial support.
a
Reaction conditions provided in Supporting Information. b Determined
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.
by thin-layer chromatography with quantification by spot densitometry (refer
to Supporting Information for additional information).
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