Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide

Article abstract of DOI:10.1021/ja045021y

Previous study showed that some Gram-negative bacteria possess human blood group activity. Among them, Escherichia coli O86 has high blood group B activity and weak blood group A activity. This is due to the cell surface O-antigen structure, which resembles that of human blood group B antigen. In this study, we sequenced the entire E. coli O86 antigen gene cluster and identified all the genes responsible for O-antigen biosynthesis by sequence comparative analysis. The blood group B-like antigen in E. coli O86 O-polysaccharide was synthesized by sequentially employing three glycosyltransferases identified in the gene cluster. More importantly, we identified a new bacterial glycosyltransferase (WbnI) equivalent to human blood group transferase B (GTB). The enzyme substrate specificity and stepwise enzymatic synthesis of blood group B-like antigen revealed that the biosynthetic pathway of B antigen is essentially the same in E. coli O86 as in humans. This new finding provides a model to study the specificity and structure relationship of blood group transferases and supports the hypothesis of anti-blood group antibody production by bacterial stimulation. Copyright

Full text of DOI:10.1021/ja045021y

Published on Web 01/27/2005
Escherichia coli O86 O-Antigen Biosynthetic Gene Cluster and Stepwise
Enzymatic Synthesis of Human Blood Group B Antigen Tetrasaccharide
Wen Yi,† Jun Shao,† Lizhi Zhu,† Mei Li,† Mamata Singh,† Yuquan Lu,† Steven Lin,† Hanfen Li,†
Kang Ryu,† Jie Shen,† Hongjie Guo,† Qingjia Yao,† C. Allen Bush,‡ and Peng G. Wang*,†
Department of Biochemistry, The Ohio State UniVersity, Columbus, Ohio 43210, and
Department of Chemistry, UniVersity of Maryland, Baltimore County, Maryland 21250
Received August 18, 2004; Revised Manuscript Received December 5, 2004; E-mail: pgwang@chemistry.ohio-state.edu
The histo-blood group ABO system is defined by ABO blood
group carbohydrate antigens, which play important roles in blood
transfusion, cell differentiation, and oncogenesis.1 The blood group
A and B antigens are formed by the transfer of two different sugar
residues (GalNAc and Gal) to the common H-disaccharide epitope
by human blood group transferases GTA and GTB, respectively.
Chemical synthesis methodology developed by Lemieux set a
milestone in the in vitro synthesis of blood group antigens.2-4 On
Figure 1. E. coli O86 O-antigen repeat unit.
the other hand, the enzymatic synthesis using recombinant GTA
and GTB has proven less laborious and more regio- and stereo-
selective.5,6 Recently, microbial glycosyltransferases have become
an attractive alternative to the mammalian counterparts, since they
are easy to express in active form without complicated gene
manipulation and can catalyze similar glycosylation reactions.
Several Gram-negative bacteria express histo-blood group anti-
gens in their lipopolysaccharide (LPS) O-chain.7,8 The sequence
of the O-antigen gene cluster facilitates the in vitro expression of
microbial glycosyltransferases for the synthesis of human antigens.
Escherichia coli O86 was previously shown to possess high blood
group B activity. Springer demonstrated that inoculation of germ-
free chicken with E. coli O86 resulted in the elevated anti-B
antibody production.9 The blood group B activity of E. coli O86
was further confirmed by an immune response on humans, and those
individuals with blood group O and A responded with a significant
rise of anti-B antibody.10 On the basis of chemical and serological
investigations, Springer et al.9 and Kochibe et al.11 showed that
the strong blood group activity of E. coli O86 came from LPS that
contained the blood group B trisaccharide partial structure. The
complete structure (Figure 1) of the O-polysaccharide12 confirmed
that its O-antigen and human blood B antigen share a similar
oligosaccharide epitope. In this communication, we sequenced E.
coli O86 O-antigen biosynthetic gene cluster and identified a new
glycosyltransferase as the equivalent of human GTB. The blood
group B antigen tetrasaccharide was synthesized in a stepwise
manner with three glycosyltransferases from the gene cluster.
A base pair sequence of 13 990 bp from E. coli O86:K62:H2
strain was obtained, which covered a continuous region from the
galF gene to the start of gnd gene (GenBank accession number
AY667408). Thirteen open reading frames were identified (Figure
2), and the respective ORFs were assigned putative functions by
sequence comparative analysis.
Figure 2. E. coli O86 O-antigen gene cluster.
Table 1. Substrate Specificity of WbnI and WbnK
WbnI
WbnK
R/Aa
(%)
R/Aa
(%)
acceptor-
acceptor
FucR-1,2Galâ-1,3GalNAc-R-OMe
Galâ-1,3GalNAc-R-OMe
FucR-1,2Galâ-OMe
100
12
30
N/A
N/A
N/A
Galâ-1,3GalNAc-R-OMe
GalR-1,3Galâ-1,4Glc
GalR-1,4Galâ-1,4Glc
Galâ-1,4Glc
Galâ-OMe
GalNAc-R-OMe
100
N/A
N/A
N/A
15
Galâ-1,4Glcâ-Oph
GalR-1,3Galâ-1,4Glc
GalR-1,4Galâ-1,4Glc
N/A
a R/A: relative activity.
cloned wbnI gene into the pET15b plasmid and expressed in E.
coli BL21 (DE3) as a His-tagged fusion protein. The sugar donor
specificity assays indicated that WbnI was capable of transferring
galactose residues at high efficiency, providing evidence that wbnI
encodes a galactosyltransferase. The acceptor screening showed the
narrow specificity of WbnI (Table 1). The activity of WbnI is nearly
7 times higher when the trisaccharide Fuc-R-1,2-Gal-â-1,3-GalNAc-
R-OMe is used as an acceptor, compared to the disaccharide Gal-
â-1,3-GalNAc-R-OMe. Thus, WbnI prefers terminal fucosylated
sugar structures to nonfucosylated ones. Interestingly, since WbnI
required R-1,2-linked fucose as a sugar side chain, the biosynthesis
of the blood group B antigen in E. coli O86 is essentially the same
as that in humans.13 The GTA and GTB are highly homologous ex-
cept for a difference in only four amino acid residues.14 The change
of four residues is sufficient to change the enzyme specificity from
using UDP-GalNAc to UDP-Gal as a sugar donor. Crystal structures
of GTA and GTB revealed that the Leu/Met 266 residue is critical
in differentiating the donors, due to the complementary size between
the amino acid residue and the functional group of the sugar donor.15
Amino acid sequence alignment of WbnI and GTB revealed that
Gln148 in WbnI was the equivalent residue of Met266 in GTB.
Gln and Met are similar in size, and preliminary modeling studies
demonstrated that Gln, in place of Met could fit well in the GTB
binding pocket. In the N-terminal of WbnI, we identified a DXD
We biochemically identified three glycosyltransferases involved
in the synthesis of blood group B antigen epitope. Orf11 (wbnI)
belongs to the glycosyltransferase family 6 with 45% similarity to
GTA and GTB. Thus, wbnI most likely encodes an R-1,3-galac-
tosyltransferase that makes the Gal-R-1,3Gal linkage to form the
blood group B-like antigen structure. To confirm its function, we
† The Ohio State University.
‡ University of Maryland.
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J. AM. CHEM. SOC. 2005, 127, 2040-2041
10.1021/ja045021y CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1 Stepwise Enzymatic Synthesis of Blood Group B
Antigen
scale by sequentially employing the three glycosyltransferases
(Scheme 1). The configuration of the anomeric carbon in starting
compound 1a was fixed as R by methyl substitution to facilitate
the NMR analysis. The products from each reaction were purified
by gel filtration, and the glycosidic linkage was analyzed by NMR
spectroscopy (see Supporting Information). The yield ranging from
75 to 93% was achieved in each single reaction, with the overall
yield of 60%. It is a substantial improvement as compared to the
chemical synthesis, for which the total yield is normally lower than
40%.
In conclusion, we elucidate the O-antigen biosynthesis in E. coli
O86, an important bacterial strain that possesses strong blood group
B activity. More importantly, we identified a new bacterial
glycosyltransferase equivalent to human blood group transferase
B (GTB). The enzyme substrate specificity and stepwise enzymatic
synthesis of blood group B antigen reveal that the biosynthetic
pathway of B antigen is essentially the same in E. coli O86 as in
humans, thus providing us an attractive way of synthesizing blood
group B antigen with microbial glycosyltransferases.
motif, which is proposed to interact with phosphate groups of the
nucleotide donor through the coordination of a metal cation. The
data therefore identify WbnI as a bacterial glycosyltranferase
equivalent to human blood group transferase GTB. Future detail
studies of WbnI can potentially reveal more insights into its
substrate specificity and structure relationships with human GTB.
The orf13 (wbnJ) was assigned as another galactosyltransferase
that transfers a Gal residue to GalNAc via a â(1f3) linkage. The
linkage specificity was confirmed by NMR analysis of the
synthesized disaccharide Gal-â-1,3-GalNAc-R-OMe (see Support-
ing Information).
Acknowledgment. We thank the National Institutes of Health
(R01 AI44040) for financial support of this work.
Supporting Information Available: Shotgun sequence of E. coli
O86 O-antigen gene cluster, cloning, expression, purification and
enzymatic assay of glycosyltransferases, stepwise enzymatic synthesis
and characterization of blood group B antigen Gal-R-1,3-(Fuc-R-1,2)-
Gal-â-1,3-GalNAc-R-OMe. This material is available free of charge
via the Internet at http://pubs.acs.org.
The C-terminal of orf14 (wbnK) has a conserved domain from
glycosyltransferase family 11, which mostly consists of R-1,2-
fucosyltransferases from different organisms. WbnK was expressed
in E. coli as a GST-tagged fusion protein. Notably, WbnK has a
strict substrate requirement shown by the acceptor specificity assays
(Table 1). The fucosyltransferase activity was identified only when
Gal-â-1,3-GalNAc (T-antigen) was used as a substrate. The change
of linkage from 1f3 to 1f4 or the configuration from â to R will
make very poor substrates for WbnK. Thus, the terminal â-1,3-
linked galactose residue is critical in WbnK recognition. Since the
substrate specificity assay reveals that WbnI has a strong preference
for a terminal fucosylated acceptor and WbnK requires a terminal
â-1,3-linked Gal sugar sequence, we can identify the biosynthetic
pathway of blood group B antigen epitope in E. coli O86. The
sequence of enzyme reactions to make blood group B antigens
in bacteria is essentially the same as in humans. This new finding
provides us not only an economical and attractive way to synthesize
human blood group antigens but also clues to elucidate the
relationship between human natural antibody production and enteric
bacterial stimulation.
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On the basis of the O-antigen structure, we carried out the
stepwise enzymatic synthesis of blood group B antigen in 10 mg
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