vaccines has succeeded in providing broad protection against infection with different serotypes and biotypes of A. pleuropneumoniae.
The most important component of current vaccines are the Apx
toxins; neutralizing antibodies raised against these major virulence
factors are effective in reducing the burden of disease but do not
prevent infection or spread of the pathogen. The recent discovery
of a highly conserved protein N-glycosylation system in
A. pleuropneumoniae represents a new target antigen with the
promise to confer broad protection [2–4].
The cytoplasmic N-glycosylation system of A. pleuropneumoniae
modifies asparagine residues of autotransporter adhesins with short
dextran oligosaccharides. The N-glycan biosynthetic pathway consists of two enzymes. The first is an asparagine glucosyltransferase
(NGT) that transfers a single glucose in β-linkage onto asparagine
residues in the Asn-X-Ser/Thr sequon (X 6 ¼ Pro). The second is an
α1,6-glucosyltransferase (α6GlcT) that synthesizes short, linear glucose oligosaccharides (oligoGlc) on the priming glucose [3]. Two
proteins modified by this system have been identified. They are
autotransporter adhesins with a high-density of target sequons [5],
suggesting that they are likely to be modified at multiple sites with
the dextran oligosaccharide. Given that the NGT and α6GlcT appear
to be constitutively expressed, that both genes for this glycosylation
system are absolutely conserved in all available genome sequences,
and that NGT activity contributes to adhesion to lung epithelial cells,
the dextran oligosaccharide may be an important and unvarying
feature of the A. pleuropneumoniae surface [4]. In this chapter, we
outline a simple method for the recombinant production of glycoconjugate virus-like particle (VLP) vaccines presenting 180 copies of
this conserved N-glycan structure on their surface.
The N-glycosylation system of A. pleuropneumoniae has been
functionally reconstituted in Escherichia coli [5]. Recombinant proteins can be targeted for glycosylation by incorporation of the
Asn-X-Ser/Thr sequon on a flexible loop or tag [6]. The VLP
scaffold that we chose for presenting the N-glycan is formed by
the coat protein from the ssRNA phage AP205 (AP205cp) [7, 8]. A
unique feature of the AP205 VLP is that the N- and C-termini of
the coat protein are exposed on the surface of the capsid and are
tolerant to genetic fusions, enabling the display of diverse peptide
and protein antigens [9]. To generate an AP205 VLP presenting
the asparagine (N)-linked dextran antigen, we genetically fused a
short peptide tag including a single glycosylation site to the
C-terminus of the AP205cp and coexpressed this construct with
NGT and α6GlcT (Fig. 1). Assembly into complete AP205 VLPs is
verified by native agarose gel electrophoresis and transmission electron microscopy. Glycosylation of the coat protein is verified by
intact protein mass spectrometry and by gel shift assays.
206
Kathryn K. Oi et al.
The most important component of current vaccines are the Apx
toxins; neutralizing antibodies raised against these major virulence
factors are effective in reducing the burden of disease but do not
prevent infection or spread of the pathogen. The recent discovery
of a highly conserved protein N-glycosylation system in
A. pleuropneumoniae represents a new target antigen with the
promise to confer broad protection [2–4].
The cytoplasmic N-glycosylation system of A. pleuropneumoniae
modifies asparagine residues of autotransporter adhesins with short
dextran oligosaccharides. The N-glycan biosynthetic pathway consists of two enzymes. The first is an asparagine glucosyltransferase
(NGT) that transfers a single glucose in β-linkage onto asparagine
residues in the Asn-X-Ser/Thr sequon (X 6 ¼ Pro). The second is an
α1,6-glucosyltransferase (α6GlcT) that synthesizes short, linear glucose oligosaccharides (oligoGlc) on the priming glucose [3]. Two
proteins modified by this system have been identified. They are
autotransporter adhesins with a high-density of target sequons [5],
suggesting that they are likely to be modified at multiple sites with
the dextran oligosaccharide. Given that the NGT and α6GlcT appear
to be constitutively expressed, that both genes for this glycosylation
system are absolutely conserved in all available genome sequences,
and that NGT activity contributes to adhesion to lung epithelial cells,
the dextran oligosaccharide may be an important and unvarying
feature of the A. pleuropneumoniae surface [4]. In this chapter, we
outline a simple method for the recombinant production of glycoconjugate virus-like particle (VLP) vaccines presenting 180 copies of
this conserved N-glycan structure on their surface.
The N-glycosylation system of A. pleuropneumoniae has been
functionally reconstituted in Escherichia coli [5]. Recombinant proteins can be targeted for glycosylation by incorporation of the
Asn-X-Ser/Thr sequon on a flexible loop or tag [6]. The VLP
scaffold that we chose for presenting the N-glycan is formed by
the coat protein from the ssRNA phage AP205 (AP205cp) [7, 8]. A
unique feature of the AP205 VLP is that the N- and C-termini of
the coat protein are exposed on the surface of the capsid and are
tolerant to genetic fusions, enabling the display of diverse peptide
and protein antigens [9]. To generate an AP205 VLP presenting
the asparagine (N)-linked dextran antigen, we genetically fused a
short peptide tag including a single glycosylation site to the
C-terminus of the AP205cp and coexpressed this construct with
NGT and α6GlcT (Fig. 1). Assembly into complete AP205 VLPs is
verified by native agarose gel electrophoresis and transmission electron microscopy. Glycosylation of the coat protein is verified by
intact protein mass spectrometry and by gel shift assays.
206
Kathryn K. Oi et al.