Glycosidase-catalysed synthesis of α-galactosyl epitopes important in xenotransplantation and toxin binding using the α-galactosidase from Penicillium multicolor

Article abstract of DOI:10.1039/a906020k

The α-galactosidase from Penicillium multicolor catalyses highly regioselective galactosyl transfer on to mono- and di-saccharide accepters that have a non-reducing terminal galactose unit to give products containing the α-D-Galp-(1→3)-D-Galp epitope found on pig tissue and which is responsible for the hyperacute rejection response in xenotransplantation of pig organs into man.

Full text of DOI:10.1039/a906020k

Glycosidase-catalysed synthesis of a-galactosyl epitopes important in
xenotransplantation and toxin binding using the a-galactosidase from
Penicillium multicolor
Suddham Singh, Michaela Scigelova and David H. G. Crout*
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL. E-mail: msrky@csv.warwick.ac.uk
Received (in Cambridge, UK) 26th July 1999, Accepted 2nd September 1999
The a-galactosidase from Penicillium multicolor catalyses
highly regioselective galactosyl transfer on to mono- and di-
saccharide acceptors that have a non-reducing terminal
galactose unit to give products containing the a-d-Galp-
oped over the last decade, requires multiple protection and
deprotection steps.10 Consequently, enzymatic synthesis is an
attractive alternative for the synthesis of oligosaccharides.
1
1
Enzymatic syntheses of a-Gal oligosaccharides have been
reported.12 However, the a-galactosyl transferase method
requires the use of a complex donor and is limited by the
availability of the appropriate enzymes.1 The use of glycosi-
dases has been reported but these have been limited by rather
low yields and by the production of mixtures.1
(
1?3)-d-Galp epitope found on pig tissue and which is
responsible for the hyperacute rejection response in xeno-
transplantation of pig organs into man.
2a
2b–d
The a-d-Galp-(1?3)-b-a-Galp moiety of glycoconjugates has
been found to be of considerable biological importance. It has
attracted attention mainly because of its significance in the
development of the xenotransplantation of animal organs into
Here we report simple, regiospecific, glycosidase-catalysed
syntheses of a-d-Galp-(1?3)-b-d-Galp-OMe and a-d-Galp-
(1?3)-a-d-Galp-OMe disaccharides and a-d-Galp-(1?3)-b-
1
human patients, a development driven on the one hand by the
d-Galp-(1?4)-d-Glcp
(1?4)-d-GlcNAcp
and
trisaccharides
a-d-Galp-(1?3)-b-d-Galp-
success of organ transplantation in the latter part of the
twentieth century and on the other by an acute shortage of donor
organs. Current research is directed towards the use of pig
organs in humans (discordant xenotransplantation), as the pig is
considered the best organ donor given the constraints imposed
by the popular opposition to the use of organs from primates.
The major problem with pig-to-man xenotransplantation is the
extremely rapid human antibody-mediated hyperacute rejection
that occurs following transplantation into human patients. The
antigenic epitopes responsible for this hyperacute rejection
response contain the a-d-Galp-(1?3)-b-d-Galp terminus and
have been identified as a-d-Galp-(1?3)-b-d-Galp-R, a-d-
Galp-(1?3)-b-d-Galp-(1?4)-b-d-Glcp-R, a-d-Galp-(1?3)-
b-d-Galp-(1?4)-b-d-GlcNAcp-R and a-d-Galp-(1?3)-b-d-
Galp-(1?4)-b-d-GlcNAcp-(1?3)-b-d-Galp-(1?4)-b-d-
catalysed by an
a-galactosidase from Penicillium multicolor discovered during
a major screening programme for synthetically useful glycosi-
dases.13 The great advantage of this enzyme is that it is highly
specific for a-(1?3) transfer and thus represents a considerable
improvement over existing methods for the synthesis of a-Gal
oligosaccharides.
Transfer of an a-galactosyl unit from p-nitrophenyl a-d-
galactopyranoside 1 on to methyl b-d-galactopyranoside 2
using an a-galactosidase from Penicillium multicolor gave only
one disaccharide a-d-Galp-(1?3)-b-d-Galp-OMe 3 in 43%
yield [Scheme 1(a)]. Similarly, transfer of an a-galactosyl unit
from donor 1 on to methyl a-d-galactopyranoside 4 gave the
disaccharide a-d-Galp-(1?3)-a-d-Galp-OMe 5 in 46% yield
[Scheme 1(b)]. These results suggest that by changing the
conformation at the anomeric position in the acceptors 2 and 4
the regioselectivity of the transfer was not affected. This
indicates that ‘anomeric control’, in which regioselectivity is
influenced by the anomeric configuration in the glycosyl
acceptor, does not operate in this system.14 In both cases the
product obtained was a (1?3)-linked disaccharide.
2
Glcp-R, known collectively as ‘a-Gal’ epitopes. These epi-
topes specifically bind to human anti-a-Gal antibodies during
3
xenotransplantation. They are found on tissues of almost all
mammals except man and old world primates. Antibodies
against the a-Gal epitope expressed in man comprise approx-
imately 1% of circulating immunoglobulin G.4 Recent in-
vestigations have shown that human anti-Gal antibodies can
Transfer of the a-galactosyl unit from donor 1 on to lactose
6 gave again the (1?3)-linked product a-d-Galp-(1?3)-b-d-
Galp-(1?4)-d-Glcp 7 in 25% yield [Scheme 1(c)]. Similarly
transfer of a-galactosyl unit from the donor 1 on to lactosamine
8 gave the trisaccharide a-d-Galp-(1?3)-b-d-Galp-(1?4)-d-
GlcNAcp 9 in 32% yield [Scheme 1(d)].
In all these reactions the conversion (donor) was 40–50%.
This was not taken into account in calculating yields. The
incomplete consumption of donor indicated product inhibition.
If this could be overcome, even higher yields should be
obtainable. We found that the released p-nitrophenol inhibited
the enzyme and showed that this inhibition could be largely
overcome by operating in less concentrated solutions. Thus by
decreasing the overall concentration of donor 1 (from 0.34 to
0.17 M) and the acceptor 8 (from 1.3 to 0.65 M), 90%
conversion was achieved and the isolated yield of the trisacchar-
ide 9 increased from 32% to 48%.
5
bind to, and be neutralised by, a-Gal-related oligosaccharides.
An a-d-Galp-(1?3)-b-d-Galp-(1?4)-b-d-GlcNAcp trisac-
charide, representing the natural a-Gal epitope, is capable of
neutralising anti-Gal antibodies at low concentrations.6
A
further reason for interest in the a-Gal epitope is that it has been
identified as a receptor for the toxin from Clostridium difficile,
a micro-organism that is a major cause of antibiotic-associated
diarrhoea and the causative agent of a serious condition,
pseudomembranous colitis, in the elderly following antibiotic
therapy.7
The possibility of utilising the adhesion of bacteria and
viruses to specific carbohydrates has become an attractive goal
in attempts to find alternatives to antibiotic therapy. Develop-
ments in this area have taken place against a background of
increasing concern about the development of antibiotic re-
sistance in important species of pathogenic bacteria.8 An
immobilised version of the C. difficile toxin receptor is in
development for the treatment of C. difficile-associated diar-
rhoea.9
The a-galactosidase used in these experiments was isolated
from Lactase P from Penicillium multicolor (KI Chemicals,
Japan). The proteins from a crude extract were precipitated by
EtOH (50% v/v) and separated by DEAE-Sepharose [equili-
brated with potassium phosphate buffer (10 mM, pH 6.5) and
eluted with a linear gradient of NaCl (0–0.8 M)] followed by
The use of a-Gal oligosaccharides for clinical applications
would require large amounts of a-Gal oligosaccharides.
Conventional synthesis of carbohydrates, although well devel-
Chem. Commun., 1999, 2065–2066
This journal is © The Royal Society of Chemistry 1999
2065

Scheme 1 Conditions: i, a-Galactosidase from Penicillium multicolor; pNP = p-nitrophenyl.
chromatography on ceramic hydroxyapatite [equilibrated with
potassium phosphate buffer (5 mM, pH 6.5) and eluted with a
linear gradient (5–400 mM) of potassium phosphate buffer (pH
3 D. K. C. Cooper, E. Koren and R. Oriol, Immunol. Rev., 1994, 141,
3
1.
4
5
U. Galili, B. A. Macher, J. Buehler and H. B. Shohet, J. Exp. Med., 1985,
1
62, 573.
6.5)]. Active fractions were concentrated using a Centriprep
P. M. Simon, F. A. Neethling, S. Taniguchi, P. L. Goode, D. Zopf,
W. W. Hancock and D. K. C. Cooper, Transplantation, 1998, 65, 346;
T. Cairns, J. Lee, L. Goldberg, T. Cook, P. Simpson, D. Spackman, A.
Palmer and D. Taube, Transplantation, 1995, 60, 1202; F. A. Neethling,
E. Koren, Y. Ye, S. V. Richards, M. Kujundzic, R. Oriol and D. K. C.
Cooper, Transplantation, 1994, 57, 959.
(
[
Amicon) membrane with a 30 kDa cut-off to give a preparation
6.4 U cm , 150 U (mg protein) ] free of b-galactosidase
2
3
21
activity.
The method described here provides a remarkably simple
one-step enzymatic synthesis of a-galactosyl epitopes and can
be scaled up for clinical applications. Given the potential
demand for a-Gal di- and tri-saccharides, the method described
here offers an inexpensive and economical route to these
compounds.
6 U. Galili and K. L. Matta, Transplantation, 1996, 62, 256.
7
G. F. Clark, H. C. Krivan, T. D. Wilkins and D. F. Smith, Arch.
Biochem. Biophys., 1987, 257, 217.
8
Towards Anti-Adhesion Therapy for Microbial Diseases, ed. I. Kahane
and I. Ofek, Plenum Press, New York and London, 1996; U. J. Nilsson,
L. D. Heerze, Y. C. Liu, G. D. Armstrong, M. M. Palcic and O.
Hindsgaul, Bioconjugate Chem., 1997, 8, 466.
In a typical experiment p-nitrophenyl a-d-galactopyranoside
1
(0.1 g, 0.332 mmol) and N-acetyllactosamine 8 (0.5 g, 1.305
mmol) in citrate–phosphate buffer (0.05 M, 2 ml, pH 4.0) were
9
L. D. Heerze, M. A. Kelm, J. A. Talbot and G. D. Armstrong, J. Infect.
Dis., 1994, 169, 1291.
incubated with the a-galactosidase from Penicillium multicolor
3
°
(
0.15 cm , 0.96 U) at 30 C for 6 h. The reaction was stopped by
10 G.-J. Boons, Tetrahedron, 1996, 52, 1095.
11 D. H. G. Crout and G. Vic, Curr. Opin. Chem. Biol., 1998, 2, 98.
heating in a boiling water bath for 5 min. Examination of the
crude product by HPLC indicated that a single trisaccharide was
formed. The product mixture was purified by carbon–Celite
chromatography15 to give trisaccharide 9 in 48% yield. The
1
2 (a) J. Fang, J. Li, X. Chen, Y. Zhang, J. Wang, Z. Guo, W. Zhang, L. Yu,
K. Brew and P. G. Wang, J. Am. Chem. Soc., 1998, 120, 6635; (b)
K. G. I. Nilsson, Tetrahedron Lett., 1997, 38, 133; (c) G. Vic, C. H.
Tran, M. Scigelova and D. H. G. Crout, Chem. Commun., 1997, 169; (d)
I. Matsuo, H. Fujimoto, M. Isomura and K. Ajisaka, Bioorg. Med.
Chem. Lett., 1997, 7, 255.
1
13
product was confirmed to be a single isomer by H and
C
NMR.
We thank the Biotechnology and Biological Sciences
Research Council (UK) for financial support and Dr K. Ajisaka
for providing the Lactase P.
1
3 M. Scigelova, S. Singh and D. H. G. Crout, J. Mol. Catal. B: Enzym.,
1999, 6, 483.
14 D. H. G. Crout, O. W. Howarth, S. Singh, B. E. P. Swoboda, P. Critchley
and W. T. Gibson, J. Chem. Soc., Chem. Commun., 1991, 1550; S.
Singh, M. Scigelova, P. Critchley and D. H. G. Crout, Carbohydr. Res.,
1
997, 305, 363.
Notes and references
1
5 W. W. Binkley, Adv. Carbohydr. Chem., 1955, 10, 55.
1
2
J. L. Platt, Nature, 1998, 392, Suppl. 11.
R. P. Rother and S. P. Squinto, Cell, 1996, 86, 185.
Communication 9/06020K
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Chem. Commun., 1999, 2065–2066