Photorhabdus luminescens lectin A (PllA): A new probe for detecting -galactoside–terminating glycoconjugates

Article abstract of DOI:10.1074/jbc.M117.812792

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for -galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting -galactosides. The -Gal epitope is present on wild-type pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA’s high specificity for -galactoside–containing ligands, and we show that PllA can be used to visualize the -Gal epitope on porcine tissues.

Full text of DOI:10.1074/jbc.M117.812792

JBC Papers in Press. Published on September 28, 2017 as Manuscript M117.812792
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.812792
Photorhabdus luminescens lectin A (PllA) - a new probe for
detecting α-galactoside-terminating glycoconjugates
a,b,c
c,d
e
e
a,b
Ghamdan Beshr , Asfandyar Sikandar , Eva-Maria Jemiller , Nikolai Klymiuk , Dirk Hauck ,
a,b
e
c,d*
a,b,c*
Stefanie Wagner , Eckhard Wolf , Jesko Koehnke , and Alexander Titz
a
Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-
6
6123 Saarbrücken, Germany
b
Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover-Braunschweig, Germany
c
Department of Pharmacy, Saarland University, Saarbrücken, Germany
d
Structural Biology of Biosynthetic Enzymes, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
D-66123 Saarbrücken, Germany
e
Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences,
LMU Munich, 81377 Munich, Germany
*
Correspondence to alexander.titz@helmholtz-hzi.de (A.T.) and jesko.koehnke@helmholtz-hzi.de (J.K.)
Keywords
Lectins, PllA, LecA, PA-IL, galactose-binding protein, α-Gal epitope
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Abstract
Lectins play important roles in infections by
pathogenic bacteria, for example, in host
colonization, persistence and biofilm formation.
Introduction
Photorhabdus luminescens (P. luminescens) is
a
Gram-negative
gamma-proteobacterium
The
bacterium
symbiotically
Gram-negative
entomopathogenic
luminescens
belonging to the Enterobacteriaceae family. In
its complex life cycle it lives symbiotically in
Photorhabdus
lives in
insect-infecting
the
intestine
of
Heterorhabditidae
nematodes and
Heterorhabditis nematodes and kills the insect
host upon invasion by the nematode. The P.
luminescens genome harbors the gene plu2096
coding for a novel lectin that we named PllA.
We analyzed the binding properties of purified
PllA with a glycan array and a binding assay in
solution. Both assays revealed a strict
specificity of PllA for α-galactoside-
terminating glycoconjugates. The crystal
structures of apo PllA and complexes with three
different ligands revealed the molecular basis
for the strict specificity of this lectin.
Furthermore, we found that a 90° twist in
subunit orientation leads to a peculiar
quaternary structure compared with that of its
ortholog LecA from Pseudomonas aeruginosa.
We also investigated the utility of PllA as a
probe for detecting α-galactosides. The α-Gal
epitope is present on wild-type pig cells and the
main reason for hyperacute organ rejection in
pig to primate xenotransplantation. We noted
that PllA specifically recognizes this epitope on
the glycan array and demonstrated that PllA can
be used as a fluorescent probe to detect this
epitope on primary porcine cells in vitro. In
summary, our biochemical and structural
analyses of the P. luminescens lectin PllA have
disclosed the structural basis for PllA’s high
entomopathogenic
pathogenically in insect larvae upon nematode
invasion. P. luminescens was first isolated in
1
977 as
a
symbiont bacterium of
Heterorhabditidae nematodes and classified
initially as Xenorhabdus luminescens (X.
1
,2
luminescens),
and later renamed P.
3
luminescens. The genus Photorhabdus consists
of the four species P. luminescens, P.
temperata, P. heterorhabditis and P.
4
-6
asymbiotica,
with the latter species being
7
pathogenic to humans. Both Xenorhabdus and
Photorhabdus species enter a wide range of
insect larvae via Steinernematidae and
4
,8
Heterorhabditidae nematodes, respectively.
Once the nematodes enter into the insect at their
infective juvenile developmental stage, their
bacterial symbionts are released into the
insect’s blood and both bacteria and nematode
9
are able to kill the larvae within 48 h.
The complete genome of P. luminescens subsp.
laumondii TTO1 was published in 2003 by
10
Duchaud et al.. Compared to other bacteria, it
shows a high number of genes predicted as
toxins and some toxins, such as Tca and Tcd,
have been extensively studied.
10-12
Often,
bacterial carbohydrate-binding proteins, i.e.
lectins, also act as toxins in addition to their
function as adhesins to enable host
colonization. A number of predicted lectins are
present in the genome of P. luminescens, but
only one example, the fucose-binding protein
specificity
for
α-galactoside–containing
ligands, and we show that PllA can be used to
visualize α-Gal epitope on porcine tissues.
1
3
PLL, has been experimentally characterized.
In Pseudomonas aeruginosa, a ubiquitous
opportunistic pathogen with a high current
Keywords
Lectins, PllA, LecA, PA-IL, galactose-binding
protein, α-Gal epitope
1
4
medical need for new therapeutics, the two
lectins LecA and LecB are both toxins and
2

adhesins with roles in biofilm formation and
In P. luminescens, the gene plu2096 was
1
5-17
18,19
persistent infection.
LecB -type proteins
previously proposed as the coding gene for a
1
0
26
are relatively common and several LecB
homologs have also been previously
characterized in other bacterial species such as
LecA-like protein and later confirmed to be
a galactose-binding lectin. An alignment of the
retrieved orthologs of LecA in each
Photorhabdus and Xenorhabdus species shows
a high degree of similarity within these
entomopathogenic species (Figure 1). While the
residues involved in metal and ligand binding
in LecA are relatively conserved, distinct
differences to P. aeruginosa LecA were
observed: LecA contains an insert flanked by
two cysteines that are absent in PllA and the
Photorhabdus and Xenorhabdus homologs
contain an additional C-terminal tail of 5 to 13
amino acids, which is partially conserved
2
0
Ralstonia
cenocepacia
violaceum.
solanaceum,
Burkholderia
Chromobacterium
2
1-23
and
2
4
The P. luminescens genome
harbors several genes of predicted lectins that
2
5
are homologs of LecB. In contrast, homologs
of LecA are comparably rare and not present in
the genomes of the former species. In P.
luminescens a single gene encoding for a LecA-
homolog (plu2096) was predicted by Duchaud
1
0
et al..
Here, we report the biochemical and structural
characterization of the P. luminescens lectin
PllA. We demonstrate that PllA is highly
specific for α-galactoside containing ligands.
By determining several crystal structures of
PllA in complex with ligands we were able to
rationalize PllA’s strong preference for α-
galactosides. We further demonstrate that PllA
can serve as a detection tool for the specific
visualization of the α-Gal epitope present on
porcine tissue. This epitope is responsible for
hyperacute rejection of pig to primate organ
xenotransplants.
(
Figure 1).
PllA has 37% sequence identity to LecA and all
amino acids whose side chains are involved in
calcium ion binding are conserved except for
one Asn (LecA) to Asp (PllA) variation. Amino
acids involved in carbohydrate recognition are
only partially conserved and three-dimensional
structural sequence variations from PllA to
LecA include Q57HQ, D103N following the
PllA amino acid numbering and a replacement
for LecA Q53 (pdb code 3ZYF) is absent in
PllA .
Recombinant production and purification of
PllA
Results and Discussion
Identification and production of PllA
The plu2096 gene was amplified from genomic
DNA of P. luminescens subsp. laumondii
TTO1 and cloned into the pET22b(+) vector.
Recombinant expression in E. coli BL21(DE3)
yielded a protein product at 13 kDa apparent
molecular weight by SDS-PAGE (Figure 2A),
which corresponds to its predicted size of 12.95
kDa. Due to the sequence homology to
galactose-binding LecA, we subjected the cell
While P. aeruginosa LecB orthologs have been
widely studied, orthologs of LecA have not
been characterized in detail. We searched the
publicly accessible NCBI database using
protein blast and the protein sequence of LecA
from strain PAO1 as a template. A moderate
number of orthologs was retrieved from only a
few different genera of Gram-negative bacteria.
2
7
lysate to galactosylated sepharose and PllA
was retained on this affinity resin. Subsequent
elution was achieved using galactose in the
These
were
mainly
entomopathogenic
Photorhabdus and Xenorhabdus species, as
well as human opportunistic pathogens from the
Enterobacter spp. and a few other pathogenic
bacterial species (see Figure S1).
elution
buffer.
Purification
yielded
approximately 6 mg of PllA per liter of
bacterial culture, which was later improved to
3

1
9 mg/L by purification on melibiose-coupled
550). This glycan structure, called the α-Gal
2
9
sepharose (see below).
epitope, is a ubiquitous constituent of glycans
in non-primate mammals and new world
monkeys. The nematode Parelaphostrongylus
tenuis also contains N-glycans decorated with
Since lectins often oligomerize and LecA forms
a tetramer, we analyzed PllA’s multimeric
state using size-exclusion chromatography
2
8
3
0
this epitope.
This epitope is mainly
(SEC). The protein’s observed apparent
responsible for hyperacute rejection of porcine
molecular weight was 27 kDa, which suggested
a PllA dimer (Figures 2B and S2). When we
used the more robust technique of dynamic
organ
xenotransplantation.
transplants29,31in
humans
Interestingly,
during
the
corresponding monovalent glycans (no. 105 and
15) showed a four- to five-fold lower binding
light scattering (DLS) we observed
a
1
homogeneous sample with an apparent
molecular weight of 52.1 kDa (Figure 2C). The
tetramer LecA was also studied by DLS under
identical conditions and its apparent molecular
weight of 52.4 kDa (Figure S3) matched the
signal (Table 1) indicating that PllA binds
carbohydrates multivalently as known for its
ortholog LecA. Interestingly, when the same
monovalent epitopes were presented on a
shorter spacer (Sp0) with one mannose between
spacer and epitope (no. 516 and 517), binding
was reduced further.
value for PllA and thus suggests
a
tetramerization of PllA in solution. The
observed differences for PllA in SEC and DLS
could result from weak interactions between
PllA and the glycan-based sepharose resin,
altering the observed molecular weight to a
smaller size.
Apart from the divalent ligands, only the two
disaccharides, Gal-α-1,3-GalNAc and Gal-α-
1,4-GlcNAc, showed a high binding signal
among the monovalent series (Figure 3A).
These two ligands are monovalently displayed
and may reveal the intrinsic specificity of PllA,
since other monovalent ligands showed only
weak or no binding to PllA on this glycan array.
Interestingly, these two ligands displayed much
stronger binding than analogous Gal-α-1,3-Gal
and Gal-α-1,2-Gal, suggesting an important role
of the acetamide moiety in the penultimate
residue for binding to PllA (Table 1). Among
the monovalent ligands on the array, the
observed linkage specificity of PllA was broad
for glycans containing terminal Gal-α-1,3- and
Gal-α-1,4-linkages. A single present Gal-α-1,6-
linked ligand (Gal-α-1,6-Glc) was moderately
bound, whereas the single Gal-α-1,2-linked
ligand (Gal-α-1,2-Gal) was not recognized by
PllA (Table 1).
Carbohydrate binding specificity of PllA
using the CFG glycan array
To assess the carbohydrate binding specificity
of PllA, the protein was fluorescently labelled
using fluoresceine isothiocyanate (FITC) and
binding to a glycan array containing over 600
carbohydrate epitopes was performed at the
Consortium for Functional Genomics (Figure
3
A). The LecA homolog PllA showed a strict
specificity towards glycans with terminal α-
galactosides, whereas β-galactosides and other
carbohydrates only showed very weak or no
binding. The highest apparent binding affinity
was detected for the bivalent α-Gal terminating
N-glycan
GlcNAc)
(Gal-α-1,3-Gal-β-1,4-
(glycans no. 360, 550)
2
Man
3
GlcNAc
2
or its difucosylated derivative glycan no. 368
bearing blood group B antigens at the non-
reducing end and the two monovalent
disaccharides Gal-α-1,3-GalNAc (glycan no.
To compare the carbohydrate specificity of
PllA with its previously characterized ortholog
3
2
LecA,
glycan array binding data of
1
12) and Gal-α-1,4-GlcNAc (glycan no. 123).
monovalent glycan ligands for both lectins was
normalized and plotted (Figure 3B). Notably,
LecA showed the best binding to Gal-α-1,4-
Gal-β-1,4-GlcNAc, which is only weakly
The highest apparent binding was observed for
bivalent Gal-α-1,3-Gal-β-1,4-GlcNAc
terminating glycans (glycans no. 360, 368,
4

recognized by PllA. This glycan is part of the
glycosphingolipid Gb3, which when bound by
LecA triggered membrane bending, a process
that was proposed as an entry pathway for P.
aeruginosa invasion into the host cell. In
contrast, PllA showed high apparent binding to
the epitopes Gal-α-1,3-GalNAc and Gal-α-1,4-
GlcNAc, whereas LecA only shows moderate
apparent binding as observed for a number of
other α-galactosides on the glycan array (Figure
the fluorescent analog 1. Among the
monosaccharides tested, free D-galactose (9),
showed inhibition of PllA with a reduced
affinity (IC50 = 1.57 mM) compared to
glycoside 12, probably due to the partial
presence of the non-binding β-anomer. Methyl
α-glycosides of D-glucose (5), L-fucose (6), D-
mannose (7) or methyl β-D-arabinoside (8) did
not show detectable binding to PllA.
Replacement of the 2-hydroxy group of
galactose with a free amine in D-galactosamine
(10) led to a two-fold increase in the binding
affinity (IC50 = 0.86 mM). Acetamide
substitution in the same position, i.e. N-acetyl-
galactosamine (11), resulted in a complete loss
of binding. Next, we tested α-galactosides with
different aglycons of varying size, e.g. methyl,
allyl, 4-nitrophenyl, 4-methylumbellifery, and
X-Gal (12-14, 16-18). These modifications only
showed a small effect on the binding affinity.
O-alkylation of ring hydroxyl groups is a
requirement for ligand binding of some lectins
3
3
3
B). In summary, LecA is a rather promiscuous
receptor for variety of monovalent
a
galactosides. In addition to PllA’s binding to
the bivalent N-glycan structures described
above, PllA was rather specific for Gal-α-1,3-
GalNAc and Gal-α-1,4-GlcNAc, one or both of
which may be the natural ligand of PllA.
Development of a competitive binding
assay for PllA
3
5,36
like the tectonins.
For PllA, O-methylation
In order to rapidly assess and quantify the
binding specificity of PllA, we developed a
competitive binding assay for PllA by utilizing
fluorescence polarization, which is based on
of the 3-hydroxyl group resulted in a complete
loss of affinity (14 → 15). A similar loss of
affinity was observed for O-methylation of the
P. aeruginosa LecB or B. cenocepacia BC2L-A
1
9,21,27,34
our previous work
for four different
2
1
ligands fucose and mannose.
lectins. Four different FITC-labelled D-
2
7
galactosides were titrated with increasing
amounts of PllA (Figure 4A-C). All three β-
linked galactosides (2, 3 and 4) did not show
binding up to 300 µM PllA. In contrast,
titration of α-linked 1 resulted in binding with a
We also tested a set of oligosaccharides
containing α-galactosyl residues for
competitive binding to PllA. Gal-α-1,3-Gal (20,
IC50 = 0.90 mM) and Gal-α-1,4-Gal (21, IC50
.08 mM) showed a two-fold lower binding
affinity to PllA than melibiose (Gal-α-1,6-Glc,
2, IC50 = 0.39 mM), whereas Gal-α-1,2-Gal
19) was only weakly active and resulted in
=
d
K of 62.7 µM as observed by the increase in
1
fluorescence polarization. These data confirmed
the strict α-galactoside specificity of PllA in
solution which was observed on the glycan
array.
2
(
approx. 50% inhibition at 10 mM. A
comparable binding specificity for Gal-α-1,6-
Glc has been reported for LecA from P.
The latter system was then used for a
competitive binding assay, where fluorescent α-
galactoside 1 was competitively displaced from
PllA using methyl α-D-galactoside (12), and a
set of available D-galactose derivatives (Figure
37
aeruginosa. The plant trisaccharide raffinose
(25) contains a terminal melibiose motif and
showed the highest binding to PllA among all
tested compounds with an IC50 of 0.11 mM.
This ubiquitous plant galactoside has also
shown an inhibitory effect on P. aeruginosa
biofilms and inhibits LecA in a similar affinity
4
D, Table 2). Interestingly, the obtained IC50
for 12 was 0.52 mM, which is approximately
0-fold weaker than the determined affinity of
1
5

3
7,38
range (K
d
= 32 µM).
The tetrasaccharide
B serotypes such as the currently used GS-IB4
isolectin from Griffonia simplicifolia.
4
0
stachyose (26) is another plant derivative of
raffinose with an additional 1,6-linked α-
galactoside moiety. For PllA an IC50 of 0.34
mM was observed, indicating that longer
oligosaccharides do not improve the binding
affinity to PllA, which is different for LecA.
Bivalent oligosaccharides containing the α-Gal
antigen Gal-α-1,3-Gal-β-1,4-GlcNAc were
identified as the apparent best ligands of the
glycan array (Figure 3A). The corresponding
monovalent trisaccharide 27 was tested in our
competitive binding assay and showed only
approx. 70% inhibition of PllA at 10 mM. This
weak binding is in good agreement with the
glycan array data where the monovalent α-Gal
epitope had reduced binding to PllA compared
to its bivalent structure (see Table 1).
PllA - a lectin with a unique tetrameric
structure
2
Apo-PllA crystallizes in spacegroup P3 21 and
crystals diffracted to 1.7 Å. Data collection and
refinement statistics for all presented PllA
structures can be found in Table 3. The core of
PllA consists of two four-stranded anti-parallel
β-sheets (Figure 5). We do not observe the
2
+
canonical Ca ion found in other C-type lectins
at the sugar binding site, which may be a result
of the crystallization buffer that contained a
high concentration of citrate known to chelate
calcium ions.
Since Gal-α-1,3-GalNAc and Gal-α-1,4-
GlcNAc were identified as the monovalent
ligands with the highest apparent affinity on the
glycan array, we tested the corresponding
biotinylated disaccharides 23 and 24,
respectively. Both soluble glycans differ only in
spacer identity and for 23 also in the anomeric
configuration from those glycans used for the
production of the glycan array. Surprisingly,
they were as active as the comparably
biotinylated α-galactosyl monosaccharide 13,
displaying IC50s from 0.59-0.66 mM.
The asymmetric unit contained two PllA
dimers, which form tetramers with symmetry
mates in accordance with DLS data. The C-
terminal 5 residue extension (Figure 1) of the
four protomers are engaged in well-defined
interactions leading to a 90° twist in the
tetramer (Figure 6A). Of the tail residues
(YSPLK), Y118 packs hydrophobically against
P120, while S119 forms two hydrogen bonds
with the tetramer partner (S119 side-chain
hydroxyl with side-chain amino group of K82,
S119 carbonyl with the main-chain of T83).
Residue L121 is inserted into a tailored
hydrophobic pocket of the tetramer partner,
which is comprised of residues L22, I28, A60,
I68, F73, I75, V79, V84, and L90 (Figure 6B).
When we compared the structure of PllA with
the structure of the well-studied protein LecA,
we found the structures to be very similar (Cα
rmsd of 0.67 over 78 atoms, Figure S11). The
main differences are found in the region
between β3 and β7 (Figure 5C), which has a
profound impact on carbohydrate binding (see
below).
3
9
1
The human blood group antigen P (28) was
also moderately recognized by PllA on the
glycan array (ligand 121, Table S1) and in the
competitive binding assay soluble monovalent
2
8 showed a moderate binding affinity (IC50
=
1
.80 mM) to this lectin. Since PllA was shown
to bind to α-Gal residues but not to GalNAc
residues (see above) and it recognized the blood
group B-terminating ligand 368 on the glycan
array (Figure 3A), we also tested soluble blood
group
B
antigens 29 and 30. These
oligosaccharides were inhibitors of PllA with
moderate potency (IC50 of 1.18 and 1.35 mM,
respectively). This specificity of PllA is in
contrast to LecA, which binds to B and A
The sequence alignment of PllA with LecA
from P. aeruginosa and the LecA homologs
from Photorhabdus and Xenorhabdus species
2
7
antigens. Thus, PllA could be used as a new
reagent for rapid identification of blood group
6

showed that PllA and LecA homologs from the
latter two species possess an extension at their
C-termini. This extension led to the surprising
result that the dimerisation of dimers is twisted
by 90° in PllA. In contrast, LecA does not
possess the 5 C-terminal residues. As a result,
the LecA tetramer is planar and formed by the
tail-to-tail arrangement of two dimers (Figure
chain of D96 and the main-chain of Y38, sugar
ring O5 with the side chain of Q57, and sugar
C6 hydroxyl with the side-chain of Q57. From
this complex structure it is clear that PllA is
only able to bind to α-galactosides: the side
chains of the two amino acids Val43 and Asn55
that are absent in LecA form a barrier and any
β-linkage would result in a clash with the PllA
surface (Figure 8). The structure also allowed
us to rationalize why D-galactosamine (10) is a
better binder than D-galactose (9), and why N-
acetyl-galactosamine (11) shows no detectable
binding: By swapping the C2 hydroxyl group
for an amino group, the sugar can now engage
in an additional hydrogen bond with the side-
chain of D103 (see Figure S8). Acetylation of
the amino-group leads to a clash with the
protein and thus abolishes binding.
6
C, 6D). We have no indication as to the
biological significance of this arrangement, but
believe that the interactions between the tail
and the tetramer partner will lead to a
significant stabilization of the tetrameric
assembly.
The structural basis of α-galactoside
specificity of PllA
To understand the α-galactoside specificity of
PllA we determined the crystal structures of
several PllA-carbohydrate complexes in the
presence of 3 mM calcium chloride in the
crystallization buffer (Figure 7): PllA in
complex with the monosaccharide methyl α-D-
galactoside (12), the trisaccharide raffinose (25)
and a FITC-labelled α-D-galactoside linked to
fluorescein (1). Complex crystals of PllA with
Complex crystals of PllA with raffinose were
also obtained by co-crystallization. The
resulting crystals belonged to space group P1
and diffracted to 1.75 Å. As expected the
overall structure of PllA does not change upon
complex formation (Cα rmsd of 0.12 Å). There
are eight protomers in the asymmetric unit and
we observed unambiguous electron density for
raffinose in all of them. Although some of the
raffinose molecules are involved in crystal
contacts, the orientation and shape of the
trisaccharide is virtually identical in each
protomer. The orientation of the galactose
moiety does not differ between the
monosaccharide and raffinose structures, and
the same hydrogen bonds are formed. The α-1,6
linkage leads glucose away from the PllA
surface, but the C4 hydroxyl forms one
hydrogen bond with the side-chain of Q57.
Interestingly, the final fructose moiety is
pointing back towards the PllA surface, giving
the raffinose an overall horseshoe shape. The
fructose C3 and C4 hydroxyls are engaged in a
hydrogen bond with the side-chain of E44. E44
is also involved in hydrogen bond formation
with the galactose C2 hydroxyl, thus linking the
two ends of the raffinose horseshoe resulting in
an additional intraligand hydrogen bond
1
2 were obtained by co-crystallization. The
resulting crystals belonged to space group P2
1
and diffracted to 1.56 Å. The overall structure
of PllA does not change upon complex
formation (Cα rmsd of 0.12 Å) and we
observed unambiguous electron density for
2
+
Ca and the ligand in each of the eight
protomers in the asymmetric unit. The
2
+
canonical Ca ion at the sugar binding site is
coordinated by the side-chains of D96, T100,
D103 and N104 as well as the main chain
carbonyl oxygen atoms of Y38 and T100. The
ligand is not involved in crystal contacts and
oriented in the same way in each protomer. It
sits in a shallow binding pocket and is engaged
in a total of 10 hydrogen bonds (protein and the
2
+
Ca ion): the anomeric oxygen (O1) and
galactoside C2 hydroxyl with the side-chain of
2
+
E44, sugar C3 hydroxyl with the Ca ion, the
side-chain of D103 and the main-chain of Y38,
2
+
sugar C4 hydroxyl with the Ca ion, the side-
7

between fructose C6 hydroxyl with galactose
C2 hydroxyl.
GlcNAc) is a ubiquitous epitope in non-primate
mammals and new world monkeys. This
carbohydrate structure is the major factor of
hyperacute rejection of xenotransplanted organs
Since we used fluorescent probe 1 in our
competitive binding assays, we wanted to
understand how the probe binds to PllA.
Complex crystals of PllA with 1 were also
obtained by co-crystallization. The resulting
2
9,41,42
in humans.
In pigs, genetic engineering
resulted in animals lacking the corresponding
galactosyltransferase thus reducing the risk of
3
1,43,44
severe immune responses.
By using
crystals belonged to space group P2
diffracted to 2.0 Å. The overall structure of the
protomers in the asymmetric unit did not
1 1 1
2 2 and
modern techniques such as CRISPR/CAS in
order to engineer animals or animal tissue
lacking the α-Gal epitope, quality controls for
the complete suppression of the biosynthetic
machineries are of crucial importance.
Currently, the isolectin GS-IB4 purified from
the plant Griffonia simplicifolia is used as a
tool to identify a wide range of α-galactoside
8
differ significantly from the apo structure (Cα
rmsd of 0.15 Å) and we observed unambiguous
density for 1 in four of the protomers. The
interactions of the galactose include all of those
observed in the other two structures. Through
fortuitous crystal packing we were able to
obtain good electron density for the fluorophore
and able to fit it. The ordered nature of the
fluorophore is the result of pi-stacking between
the tricyclic ring systems of two molecules of 1
bound to symmetry mates.
4
5
epitopes, among which is the α-Gal epitope.
Due to the high selectivity of PllA, this
bacterial lectin could be an alternative to the
currently used GS-IB4.
The crystal structure of GS-IB4 in complex
with the terminal disaccharide Gal-α-1,3-Gal
(20) as a methyl glycoside shows extensive
interactions between the terminal galactose
residue and the protein but no contacts with the
reducing end galactose moiety. To compare
the recognition features of both proteins to this
epitope, we have docked the methyl glycoside
of 20 into the carbohydrate binding site of PllA
The strict specificity towards α-galactosides is
unique for PllA when compared to LecA. From
the crystal structure of PllA with 12, it becomes
clear that β-galactosides cannot be recognized
without a steric clash with the protein surface of
PllA (Figure 8A). In contrast, LecA opens a
shallow cleft close to its anomeric center that
allows the accommodation of large β-linked
aglycons, such as in 4-nitrophenyl β-D-
galactoside. From a superposition of the
binding site amino acid residues of PllA with
LecA, it can be deduced that the additional
amino acids Val43 and Asn55 present in PllA
are responsible for preventing PllA from
binding β-galactosides (Figure 8B).
4
6
(Figure S9). In this computed structure, the
terminal saccharide moiety forms extensive
contacts with the lectin receptor. In contrast to
GS-IB4, the reducing end galactose established
two more hydrogen bonds with E44 and N55 of
PllA, which may serve as an explanation for the
high specificity of PllA for the Gal-α-1,3-Gal
epitope.
We thus tested the suitability of PllA to detect
the α-Gal epitope in wild-type primary pig
kidney cells and cells derived from the
Application of PllA for the detection of the
corresponding
α-1,3-galactosyltransferase
α-Gal epitope
knockout (GTKO) animals (Figure 9). Both,
PllA and the current standard lectin GS-IB4
visualized the α-Gal antigen in wild-type
porcine cells similarly. Since the α-Gal antigen
is also present on glycolipids in red blood
PllA showed the highest apparent binding on
the glycan array to a biantennary N-glycan
structure carrying the α-Gal epitope on its
antenna. This antigen (Gal-α-1,3-Gal-β-1,4-
4
1
cells,
we performed hemagglutination
8

experiments with red blood cells (RBC) from
wild-type and GTKO pigs. PllA agglutinated
wild-type porcine red blood cells but was
unable to agglutinate RBCs from the GTKO pig
glycoproteins of the nematode Haemonchus
contortus and vaccination of lambs with
glycoproteins of the sheep parasite H. contortus
specifically elicited anti-Gal-α-1,3-GalNAc IgG
antibodies. In addition, α-linked galactosyl
residues have been identified in C. elegans N-
4
9
(Figure 10A, B). In addition, we could further
show that this agglutination was galactose-
dependent and could be inhibited by the
presence of raffinose (Figure 10C).
5
0,51
glycans attached to mannose residues
or
5
2
attached to core fucose residues. Importantly,
the nematodes C. elegans and H. contortus are
both phylogenetically closely related to
Heterorhabditis, whereas A. suum is more
Conclusion
53
distantly related. It is thus reasonable to
speculate that the Gal-α-1,3-GalNAc epitope
recognized by the bacterial lectin PllA, is also
present in the nematode Heterorhabditis and
plays a role in bacterial attachment or
symbiosis of Photorhabdus species with their
native nematode hosts. Moreover, this epitope
has been described as one terminal constituent
of glycosphingolipids of the insect Calliphora
vicina pupae and members of the order of
diptera, i.e. flies, are generally susceptible to
infection with Heterorhabditis and P.
luminescens. Therefore, it is possible that Gal-
α-1,3-GalNAc is one natural ligand bound by
PllA both in the nematode symbiont and in
infected insects.
The opportunistic pathogen P. aeruginosa
utilizes the two soluble lectins LecA and LecB
for infection of the host and biofilm formation.
While numerous LecB-like proteins have been
characterized, LecA orthologs are scarce. Here,
we show that various orthologs of LecA are
present in the insect pathogenic bacteria from
the Photorhabdus and Xenorhabdus species as
well as in the human gut bacterium and
pathogen Enterobacter spp.. A high degree of
similarity was observed among those orthologs
with LecA having a sequence insert and lacking
an otherwise conserved C-terminal tail.
5
4
The gene plu2096 from the entomopathogenic
bacterium P. luminescens was cloned and
recombinantly produced in high production
yields. It encodes the galactose-binding lectin
PllA with 37% identity to LecA. The
carbohydrate binding specificity of PllA was
assessed on a glycan array containing over 600
different carbohydrate epitopes. Interestingly,
PllA showed very strict specificity toward α-
galactosides with high apparent binding to the
α-Gal epitope as well as to Gal-α-1,4-GlcNAc
and Gal-α-1,3-GalNAc.
Based on the carbohydrate specificity of PllA
as determined by the glycan array, PllA was
tested in a porcine cell culture staining
experiment for the detection of the α-Gal
epitope, the prime reason for hyperacute organ
rejection in xenobiotic transplants. PllA proved
to be a suitable detection tool and specifically
detected the α-Gal epitope in porcine tissue and
on red blood cells. This fact qualifies
recombinantly produced PllA for the efficacy
assessment of methods to genetically
manipulate cells, such as CRISPR/CAS, for the
production of alternative animal cells, tissue or
organisms lacking the α-Gal epitope as donors
for xenotransplantation.
To date, the biological role of the Gal-α-1,4-
GlcNAc epitope remains unclear and natural
sources have not been identified despite the fact
that anti-Gal-α-1,4-GlcNAc antibodies are
4
7
present in human serum. In contrast, Gal-α-
,3-GalNAc epitopes are present in nematodes,
We are currently analyzing the biological role
of PllA in P. luminescens for its life cycle in
nematodes and insects. Furthermore, it will be
of interest to analyze the role of PllA orthologs
in the human pathogens P. asymbiotica and the
1
and have for example been described in
glycolipids from the worms Ascaris suum and
4
8
Caenorhabditis elegans. Furthermore, Gal-α-
,3-GalNAc is a ubiquitous epitope present on
1
9

Enterobacter spp., a group of bacteria that are
part of the normal human gut flora with
pathogenic potential.
3.76 (m, 2H, H2, H3), 3.73 - 3.69 (m, 2H, H6).
1
3
C NMR (101 MHz, MeOH-d4) δ 135.65
(CH CHCH O-), 117.48 (CH CHCH O-),
2
2
2
2
9
9.46 (C1), 72.37 (C5), 71.51 (C2/3), 71.16
(
C4), 70.21 (C2/3), 69.39 (CH CHCH O-),
2
2
6
2.78 (C6).
Materials and methods
Chemicals
Allyl
3-O-methyl-α-D-galactopyranoside
Methyl α-L-fucoside (6), methyl α-D-mannoside
(15). A microwave vial was filled with allyl
galactoside 14 (57 mg, 0.26 mmol) and
dibutyltinoxide (71 mg, 0.29 mmol) and the
reagents were dried in vacuo. Dry PhMe/MeCN
(5:1, 660 µL) was added and the suspension in
the sealed tube was exposed to microwave
irradiation for 20 minutes at 150 °C. The clear
solution was allowed to cool to 50 °C and to the
resulting suspension was added MeI (405 µL,
6.5 mmol) drop-wise. The reaction was stirred
for 48 h at 50 °C, after removal of the volatiles
in vacuo and purification of the crude product
by MPLC, the title compound was obtained as
(7), D-galactose (9) were purchased from
Dextra Laboratories (Reading, UK); D-
galactosamine (10), methyl α-D-galactoside
(12), p-nitrophenyl α-D-galactoside (16), 4-
methylumbelliferyl α-D-galactoside (17), 5-
bromo-4-chloro-3-indolyl α-D-galactoside (18),
isopropyl β-D-1-thiogalactoside (IPTG) from
Carbosynth Ltd. (UK); methyl β-D-arabinoside
(8) from Tokyo Chemical Industry (Japan);
methyl α-D-glucoside (5), N-acetyl-D-
galactosamine (11), stachyose (26) from Sigma
Aldrich (Germany); melibiose (22) from MP
Biomedicals LLc. (France); raffinose (25) from
Th. Geyer Laboratories (Germany); Gal-α-1,3-
Gal (20), Gal-α-1,4-Gal (21), Xeno antigen
1
pure product (40 mg, 65%). H NMR (400
MHz, MeOH-d4) δ 6.08 – 5.89 (m, 1H,
CH
CH
CH
2
2
2
CHCH
CHCH
CHCH
2
2
2
O-), 5.34 (dd, J = 17.2, 1.8 Hz, 1H,
O-), 5.17 (dd, J = 10.4, 1.6 Hz, 1H,
O-), 4.85 (d, J = 4.0 Hz, 1H, H1),
(
(
27), P1 antigen (28), blood group B antigens
29, 30) from Elicityl OligoTech (France); Gal-
α-SP-Biotin (13), Gal-α-1,3-GalNAc-α-SP-
Biotin (23), Gal-α-1,4-GlcNAc-β-SP-Biotin
4.23 (ddt, J = 13.0, 5.3, 1.6 Hz, 1H, 1H,
CH CHCH O-), 4.13 (dd, J = 3.3, 1.2 Hz, 1H,
H4), 4.04 (ddt, J = 13.0, 6.1, 1.4 Hz, 1H,
CH CHCH O-), 3.85 (dd, J = 10.1, 3.9 Hz, 1H,
H2), 3.82 – 3.77 (m, 1H, H5), 3.76 - 3.63 (m,
2H, H6), 3.46 (s, 3H, CH ), 3.42 (dd, J = 10.1,
.2 Hz, 1H, H3). C NMR (101 MHz, MeOH-
d4) 135.65 (CH CHCH O-), 117.50
CHCH O-), 99.37 (C1), 81.15 (C3), 72.35
C5), 69.38 (CH CHCH O-), 69.22 (C2), 66.91
C4), 62.74 (C6), 57.24 (CH ).
2
2
(
1
(
24) from Lectinity (Russia); Gal-α-1,2-Gal-β-
-OMe (19) from Carbohydrate Synthesis
Oxford, UK). Fluorescent ligands 1-4 were
2
2
2
7
synthesized as described.
3
1
3
3
δ
2
2
Allyl α-D-galactopyranoside (14) was
synthesized from galactose in a Fischer-type
glycosylation with allyl alcohol in presence of
(
(
(
CH
2
2
2
2
3
+
Amberlite IR120/H . The title compound was
1
obtained by recrystallization. H NMR (400
MHz, MeOH-d4) δ 5.98 (dddd, J = 17.2, 10.4,
Bioinformatics
6
1
1
3
.1, 5.2 Hz, 1H, CH
7.2, 1.7 Hz, 1H, CH
0.4, 1.4 Hz, 1H, CH
.0 Hz, 1H, H1), 4.23 (ddt, J = 13.0, 5.3, 1.5
CHCH O-), 4.04 (ddt, J = 13.0,
.1, 1.4 Hz, 1H, CH CHCH O-), 3.93 – 3.89
m, 1H, H4), 3.85 - 3.78 (m, 1H, H5), 3.78 -
2
CHCH
CHCH
CHCH
2
O-), 5.33 (dq, J =
O-), 5.17 (dq, J =
O-), 4.87 (d, J =
2
2
A BLAST search (blastp) was done using the
amino acid sequences of LecA from P.
aeruginosa as query (accession number
Q05097). The search was carried out choosing
non-redundant protein sequences database with
exclusion of P. aeruginosa (taxid:287). The
2
2
Hz, 1H, CH
2
2
6
2
2
(
1
0

best 100 matches were chosen for an alignment
terminator (5’ GCTAGTTATTGCTCAGCGG
3’).
5
5
using the COBALT tool.
The aligned
sequences were clustered using the SECATOR
5
6
57
For expression, pET22b-pllA was transformed
into chemically competent E. coli BL21(DE3)
and the expression strain was selected on LB
agar supplemented with ampicillin (100 µg
algorithm, which relies on BIONJ to build
the phylogenetic tree. The best LecA-like
sequences (with lowest E-value) from each
Xenorhabdus and Photorhabdus species were
aligned with LecA using COBALT and
conserved sequence was colored using Color
−
1
mL ). 2 L of LB supplemented with ampicillin
−
1
(
100 µg mL ) were inoculated with a
5
8
preculture and grown at 37 °C and 180 rpm to
an OD600 of 0.5-0.6. Expression was induced
with addition of IPTG (0.5 mM final
concentration) and bacteria were then further
cultured for 6 h at 30 °C and 180 rpm. The cells
were harvested by centrifugation (3000 x g, 10
min) and the pellet was washed with TBS/Ca
Align Conservation. The amino acid sequence
of hypothetical LecA homologs (with lowest E-
value in Xenorhabdus and Photorhabdus
species)
luminescens),
are
WP_011146351.1
WP_046975865.1
(P.
(P.
temperata), WP_065824676.1 (P. asymbiotica),
WP_054480913.1
WP_038256436.1
WP_013184196.1
WP_047963870.1
WP_074019816.1
(P.
heterorhabditis),
bovienii),
nematophila),
khoisanae),
thuongxuanensis),
(
20 mM Tris, 137 mM NaCl, 2.6 mM KCl at
pH 7.4 supplemented with 100 µM CaCl ). The
cells were resuspended in 25 mL TBS/Ca with
(X.
(X.
(X.
2
−
1
PMSF (1 mM) and lysozyme (0.4 mg mL )
and subsequently disrupted by 5 cycles in a
microfluidics homogenizer (Microfluidics
Corp., USA). Cell debris was removed by
centrifugation (10’000 x g, 60 min) and the
supernatant was loaded onto a column
(X.
WP_038237499.1 (X. szentirmaii), and
GenBank number SFO04414.1 (X. japonica),
SFJ01328.1 (X. mauleonii).
5
9
containing galactosylated or later melibiose-
coupled sepharose CL-6B. The column was
washed with TBS/Ca and PllA was eluted by
addition of 100 mM galactose or 100 mM
raffinose to the buffer. The eluted fractions
were extensively dialyzed against distilled
water and then TBS/Ca buffer. The
concentration was determined by UV
absorbance at 280 nm using a calculated molar
extinction coefficient of 19’480 M cm . The
yield of purified PllA was 6 mg (galactose-
column) or 19 mg (melibiose-column) per L
culture volume.
Cloning, expression and purification of
recombinant PllA
Genomic DNA was isolated from P.
luminescens subsp. laumondii TTO1 using
GenElute Bacterial Genomic DNA Kit (Sigma
Aldrich). The plu2096 gene sequence was
amplified by PCR with Phusion polymerase
(
New England Biolabs, UK) and primers
introducing NdeI (5’
GGAATTCCATATGTCTGATTGGTCAGGA
AG 3’) and BamHI (5’
−1
−1
CGGGATCCTTATTTTAAAGGGGAGTATC
GAG 3’) restriction sites. After digestion of the
expression vector pET22b(+) (Novagen,
Germany) and the PCR product with NdeI and
BamHI (New England Biolabs, UK), ligation of
the insert was performed with T4 DNA ligase
Gel filtration
A HiLoad 16/600 Superdex 200 pg (GE
Healthcare) equilibrated with TBS/Ca buffer
(New England Biolabs, UK) resulting in
(
20 mM Tris, 137 mM NaCl, 2.6 mM KCl at
pH 7.4 supplemented with mM
CaCl ) with flow rate of mL/ min. A
plasmid pET22b-pllA. The sequence was
confirmed by sequencing (GATC Biotech,
Germany) with primers T7 promotor (5’
TAATACGACTCACTATATAGG 3’) and T7
1
2
1
calibration curve for molecular size estimation
was generated by loading 10 µM
1
1

of mixture of standard proteins
(lysozyme,
Direct binding of fluorescent ligands 1-4
to PllA
DNase I, ovalbumin and BSA). Thereafter, 10
µM of PllA was loaded on the column and
analyzed with the same flow rate.
1
(
3
0 µL of a serial dilution of PllA in TBS/Ca
618 - 0.30 µM) was added in triplicates to a
84-well plate (Greiner Bio-One, Germany, cat
no 781900). Then, 10 µL of fluorescent ligand
- 4 dissolved in TBS/Ca, were added to PllA
Dynamic
light
scattering
(DLS)
1
measurements
to a final concentration of 10 nM. After
incubation for 1 h at r.t., blank corrected
fluorescence intensity was recorded using a
PheraStar FS microplate reader (BMG Labtech
GmbH, Germany) with excitation filters at 485
nm and emission filters at 535 nm and
fluorescence polarization was calculated. The
data were analyzed using a four parameter fit of
the MARS Data Analysis Software (BMG
Labtech GmbH, Germany). A minimum of
three independent experiments on three plates
was performed for each fluorescent ligand.
DLS measurements were performed on a
Zetasizer Nano-ZS (Malvern Instruments, UK).
Stock solutions were filtered with a syringe
filter before measurements. 50 µL of PllA or
LecA (100 mM) in TBS/Ca (20 mM Tris, 137
mM NaCl, 2.6 mM KCl at pH 7.4
supplemented with 1 mM CaCl
at 25 °C.
2
) was measured
Fluorescent labelling of PllA and glycan
array analysis
Competitive binding assay for PllA
2 3
PllA (700 µL, 58 µM in Na CO buffer, pH 9.3)
was incubated at r.t. under shaking (500 rpm)
1
0 µL of a serial dilution of each tested
with fluorescein isothiocyanate (FITC, 33 µL, 3
compounds in TBS/Ca (20 - 0.01 mM) were
added in triplicates to a 384-well plate (Greiner
Bio-One, Germany, cat no 781900).
Afterwards, 10 µL of PllA and 1 were added to
each well at final concentrations of 55 µM and
-
1
mg mL , in sodium carbonate buffer, pH 9.3)
for 1 h. Purification of the labelled protein was
performed as described above for unlabelled
PllA, the protein concentration was determined
1
9
as previously described for LecB-PA14 using
1
0 nM, respectively. After incubation for 1 h at
−
1
−1
an extinction coefficient of 19’480 M ·cm
r.t., fluorescence polarization was determined
using a microplate reader as described above.
The data were analyzed using a four parameter
fit of the MARS Data Analysis Software (BMG
Labtech GmbH, Germany). A minimum of
three independent experiments on three plates
was performed for each compound.
for PllA.
FITC-labeled PllA was tested on the
Consortium for Functional Glycomics (CFG)
mammalian glycan array (Core H) version 5.3
containing 600 printed glycans in replicates of
6
. Standard procedures of Core H (details see
http://www.functionalglycomics.org/glycomics/
publicdata/selectedScreens.jsp) were run at 5
-
1
-1
µg mL , and 50 µg mL protein based on the
X-ray crystallography
6
0
protocol by Blixt et al.. Raw data of the PllA
binding experiments are available as
Supporting Table S1 and S2 as an XLS
spreadsheet.
Crystals of apo-PIIA were obtained in 1.6 M
sodium citrate tribasic dihydrate pH 6.5. To
solve the PIIA-ligand complex structures, PIIA
was co-crystallized in the presence of 10 mM
ligand and 3 mM calcium chloride. Optimized
crystals of PIIA-12, PIIA-25 and PIIA-1 were
1
2

grown under conditions of 0.2 M ammonium
acetate, 0.1 M Bis-Tris buffer pH 5.5 and 25%
PEG 3350; 0.2 M magnesium acetate and 20%
PEG 3350, and 0.15 M DL-malic acid and 20%
PEG 3350, respectively. Diffraction data for all
proteins was collected from single crystals at
Pigs lacking the α-1,3-galactosyl-galactose
epitope (GTKO) were generated by disrupting
the causative galactosyl transferase gene
GGTA1, according to the procedure described
6
9
in Klymiuk et al.. First, a bacterial artificial
chromosome (BAC) containing the target
region of the porcine genome, CH242-21F3,
was modified by bacterial recombineering in a
way that it contained a STOP box right after the
START codon of GGTA1, resulting in the
termination of protein translation as well as
RNA transcription of the gene, as well as a
floxed resistance cassette for neomycin
selection. Then, this modified BAC was
nucleofected into pig primary cells according to
1
00 K. Data for apo-PIIA and PllA-12 was
obtained at beamline ID23-2 (ESRF) at a
wavelength of 0.873 Å, while data for the PIIA-
2
5 and PIIA-1 was collected at beamline ID30-
B (ESRF) at a wavelength of 0.967 Å. Data was
6
1
62
processed using Xia2 or XDS and the
6
3
structures solved using PHASER Molecular
Replacement with LecA (PDB ID 1L7L) as a
search model. The models were manually
6
4
70
rebuilt with COOT
and refined using
Richter et al. and single cell clones were
6
5
66
PHENIX and Refmac5. The structures were
generated under antibiotic selection and
propagated to yield cells for DNA isolation and
somatic cell nuclear transfer (SCNT). Single
cell clones were screened for homologous
recombination by a qPCR-based loss-of-wild-
type-allele approach and cell clones that
indicated a heterozygous modification of the
GGTA1 allele were used for SCNT to generate
heterozygous knockout pigs. After birth, one of
the animals was sacrificed and primary cells
were cultivated and nucleofected with a
plasmid encoding Cre recombinase. Again,
single cell clones were generated and now
screened for the removal of the neomycin
selection cassette. Another round of SCNT was
performed to generate heterozygous KO
animals lacking the neomycin selection
cassette. Pigs were then maintained and bred to
achieve homozygous GTKO pigs after two
generations. Primary cells from such GTKO
animals were isolated according to the
validated using MolProbity and all images were
created using PyMOL.
6
7
Molecular Modeling
Docking was performed using PLANTS v1.1.
6
8
The calculation of charge and energy
minimization of the protein and ligand was
performed
with
Molecular
Operating
Environment (MOE) v. 2014.09 (Chemical
Computing Group Inc., 1010 Sherbooke St.
West, Suite #910, Montreal, QC, Canada, H3A
2
R7, 2015). Then, the standard docking
procedure (PLANTS manual available at
http://www.tcd.uni-
konstanz.de/plants_download/download/manua
l1.1.pdf) was used to dock D-galactosamine
(10) and the methyl glycoside of 20 into the
binding pocket of the apo-PllA crystal
structure. The docking site was limited to a 13
Å radius sphere centered in the mass center
7
0
procedure described by Richter et al. and
these cells were used for evaluating the
specificity and sensitivity of the PllA lectin.
(coordination: X = -8.624, Y = 15.131 and Z =
4
5.115) of the crystallized protein. Asp103,
Asp96, Gln57 and Glu44 were set as flexible
residues in the input file.
Lectin staining of porcine cells
4
For lectin staining, 1x10 cells were seeded in
6
-channel
slides
(IBIDI,
Martinsried,
Generation of primary GTKO cells
Germany), coated with collagen type 1 (Serva
Electrophoresis, Heidelberg, Germany), and
1
3

7
0
cultivated under conventional conditions.
times. A 10% pRBC solution was prepared by
diluting 1 mL pRBCs with 9 mL PBS (OD600
= 7). Then, 50 µL PBS was added to each well
of a 96-well plate. Thereafter, 50 µL of PIIA
(2.5 mg/mL) were added to the first well, mixed
and 50 µL of this mixture was transferred to the
second well. Serial dilution of PIIA was
continued until 23 dilutions were obtained. 50
µL 10% pRBCs from WT or GTKO pig were
added to each well and the plate was incubated
When reaching a confluence of 80-100%, cells
were stained for 15 min with 5 µg/mL Hoechst
3342 and subsequently with FITC-labelled
isolectin B4 (GS-IB4, Sigma, 500 µg/mL) or
FITC-labelled PllA (50 µg/mL), for 1 hour at
room temperature. After washing with PBS,
cells were visualized in a fluorescence
microscope (Axiovert 200, Zeiss).
3
2
h at room temperature. Inhibition of PllA-
mediated WT pRBC agglutination was then
tested with raffinose. A serial dilution of
raffinose (20 mM) was mixed with the lowest
lectin concentration showing agglutination (2.4
µg/mL). After incubation for 30 min at r.t.
pRBCs were added. The plate was incubated
for 2h at r.t..
Hemagglutination of porcine red blood
cells
Hemagglutination was done in analogy to a
3
4
previously published protocol.
Lithium-
Heparinized pig blood was centrifuged at 1’000
x g for 5 min. Plasma was removed and the
pRBCs were washed with 45 mL PBS for 3
1
4

Acknowledgements
The authors are grateful to Prof. Rolf Müller (HIPS Saarbrücken) for providing the P. luminescens
strain used in this study and to the Consortium for Functional Glycomics (Core H, Protein-glycan
Interaction Resource of the CFG and the supporting grant R24 GM098791) for the glycan array
analysis of PllA. We acknowledge use of the ESRF synchrotron (beamlines ID23-2 and ID30-B).
GTKO pigs were produced with funding from DFG TRR 127 ‘Biology of xenogeneic cell, tissue, and
organ transplantation - from bench to bedside’. J.K. would like to thank the Deutsche
Forschungsgemeinschaft for an Emmy Noether Fellowship (KO4116/3-1). A.T. acknowledges the
Helmholtz-Association (grant no. VH-NG-934) and the Deutsche Forschungsgemeinschaft (grant no.
Ti 756/2-1) for generous funding.
Author contributions: AT conceived and coordinated the study. GB, JK and AT wrote the paper. GB
and AT designed, performed and analyzed the experiments shown in Figures 1-4, Tables 1-2. GB
performed docking experiments. DH performed chemical synthesis of compounds. SW designed and
performed cloning of PllA expression vector with assistance of GB. AS and JK designed, performed
and analyzed the experiments shown in Figures 5-8, Table 3 with assistance of GB. EMJ, NK and EW
designed, performed and analyzed the experiments shown in Figure 9-10. All authors reviewed the
results and approved the final version of the manuscript.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
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2
0

Table 1. Comparison between high binding glycans and structurally related glycans on the array, i.e. comparison of linkage-
types and multivalent-monovalent effect. Note: spacers vary on the glycan array.
Table 2. Evaluation of natural and synthetic inhibitors of PllA using the competitive binding assay. Averages and standard
deviations were obtained from three independent experiments. n.i.: no inhibition observed up to 10 mM. SP: spacer -
(
2 3 2 5
CH ) NH-CO(CH ) NH- .
Table 3. Data collection and refinement statistics. Statistics for the highest-resolution shell are shown in parentheses.
Figure 1. Sequence alignment of LecA from P. aeruginosa with hypothetical LecA-like proteins from Photorhabdus and
Xenorhabdus species (one single ortholog per organism selected based on highest identity to LecA). Strictly conserved
amino acids are shaded black and similar conserved amino acids are shaded grey. Black dot: amino acid of LecA involved
2
+
in Ca binding; black triangles: amino acids of LecA involved in sugar binding; asterisks: amino acids of LecA involved in
2
+
both Ca and sugar binding. Amino acid numbering follows the LecA crystal structure where the N-terminal methionine is
lacking. The depicted protein sequence of P. luminescens (PllA) is encoded by the plu2096 gene.
Figure 2. A Recombinant expression and affinity purification of PllA analyzed by SDS-PAGE (15%). E. coli whole cell
extracts of uninduced (lane 1) and IPTG-induced cultures (lane 3), and purified PllA (lane 4), molecular weight marker in
kDa (lane 2); B sepharose size exclusion chromatogram of PllA; C DLS analysis of PllA.
Figure 3. Carbohydrate specificity of PllA on a glycan array. A Profiling of the glycan binding specificity of PllA on a
glycan array. Glycans containing terminal α-galactoside are colored in black, those with terminal β-galactoside in red and
oligosaccharides with neither of these terminal moieties are colored in blue. Selected structures showing highest apparent
binding are illustrated in CFG notation. B Comparison of glycan binding specificity between PllA (this work) and LecA
3
2
(
PA-IL ) from Pseudomonas aeruginosa based on normalized CFG glycan array data. Normalization by dividing RFU
averages of the glycan of interest by the RFU average of the ligand with highest apparent affinity on the respective array
i.e., glycan 550 for PllA, glycan 31 for LecA). Glycan structures are indicated and depicted with the different spacers of the
(
array for LecA. For the PllA array, identical glycans are generally contain β-Sp8 spacers except 105 (Sp0) and 116 (β-Sp0).
Numbers on top of columns indicate glycan number of the respective arrays. For A and B, error bars correspond to 6
replicates.
Figure 4. Establishing a carbohydrate binding assay for PllA in solution. A Structure of fluorescent ligands 1-4 based on D-
galactose. B Titration of fluorescent ligands 1-4 with PllA. C Dissociation constant for 1 was obtained from a four
parameter fitting procedure to the dose-dependent increase in fluorescence polarization (K
inhibition of the binding of 1 to PllA with methyl α-D-galactoside (12, IC50 = 0.52 ± 0.07 mM) and raffinose (25, IC50
.11 ± 0.01 mM). One representative titration experiment of triplicates on one plate is shown. Dissociation constant and
standard deviations given were obtained from at least three independent replicates of triplicates on three plates each.
d
62.7 ± 3.8 µM). D Competitive
=
0
Figure 5. A Sequence of PllA. Secondary structure elements are shown above the sequence (blue arrows: β-strands, red
2
+
barrel: α-helix). Residues responsible for sugar binding are highlighted with magenta stars, Ca -binding residues with cyan
circles and amino acids coordinating both as yellow triangles. Tail residues unique to PllA and its close homologs are
highlighted with a blue box. B Cartoon representation of a PllA apo monomer. C Fold diagram for the structure shown in B.
2
1

Figure 6. Overall structure of PllA and comparison to LecA. A Cartoon representation of the PllA tetramer. Two parallel
dimers (yellow/magenta and green/cyan) form tail-to-tail dimers with a 90° twist. B Detailed view of the PllA tail-to-tail
interface. We observe two hydrogen bonds between the side-chains of tail S119 (cyan) and K82 (yellow) and the C-
terminus of the tail and the backbone nitrogen of A74 (yellow). In addition, tail residue L121 is inserted into a hydrophobic
pocket of its binding partner. C The LecA tetramer is planar, formed by tail-to-tail dimerization of two parallel dimers
(
yellow/magenta and green/cyan). D The much shorter tail of LecA provides several stabilizing hydrogen bonds (dashed
lines), but the interactions are not sufficient to cause a twist of the two dimers relative to each other.
Figure 7. PllA - carbohydrate complex structures. A, B PllA bound to methyl α-D-galactoside (12). This interaction is
stabilized through ten hydrogen bonds (dashed lines). Eight of them are between the ligand an the protein, while two are
2
+
provided by the Ca ion. C, D PllA bound to raffinose (25). In addition to the hydrogen bonds observed in A, the glucose
moiety forms a hydrogen bond with the side-chain of Q57, while terminal fructofuranoside forms two hydrogen bonds with
the side-chain of E44, which results in the ligand adopting a horseshoe shape. E, F PllA bound to fluorescent tracer 1. No
interactions with the protein are observed beyond the carbohydrate moiety. The fluorescein can only be observed as the
result of fortuitous crystal contacts in half of the monomers in the asymmetric unit. PllA is shown as a yellow
2
+
cartoon/surface representation, ligand as grey sticks, oxygen atoms red, nitrogen atoms blue, sulfur atoms yellow, and Ca
ions as green spheres. Difference electron density (F0-Fc) contoured to 3σ with phases calculated from a model that was
refined in the absence of metal ions is shown as grey isomesh (B, D, F).
Figure 8. Rationalizing PllA α-galactoside specificity. A Representation of the PllA (yellow) binding pocket with methyl α-
D-galactoside (12, grey sticks). 4-nitrophenyl β-D-galactoside (salmon sticks and isomesh, taken from PDB 3ZYF) was
superposed onto α-D-galactoside. Due to the restricted ligand binding site of PllA only α-substituted ligands, leading away
from the surface, can be accommodated while β-substituted ligands clash. B Superposition of the binding site amino acid
residues of PllA (blue) with LecA (magenta), oxygen atoms red, nitrogen atoms blue. Residue numbers correspond to PllA.
Figure 9. Staining of primary porcine kidney cells from wild-type pigs (WT) and GGTA1 KO (GTKO) animals unable to
produce the α-Gal antigen. Fluorescein-tagged PllA or GS-IB4 were used as probes and detected the α-Gal antigen in WT
cells. Lectin concentration: PllA, 50 µg/mL; GS-IB4, 500 µg/mL, 400x magnification.
Figure 10. Hemagglutination of porcine red blood cells by PllA. A wild-type pig RBCs. B GTKO pig RBCs. C Inhibition
of PllA-mediated agglutination of wild-type pig RBCs with raffinose.
2
2

Table 1.
2
3

Table 2.
2
4

Table 3.
PllA, apo
PllA, Me-α-Gal (12) PllA, Raffinose (25) PllA, fluorescent
ligand 1
5
OFZ
5ODU
5OFX
5OFI
Resolution range
46.05 - 1.75 (1.813 -
47.2 - 1.56 (1.616 - 44.09 - 1.75 (1.813 -
43.76 - 2.0 (2.071 -
1
.75)
1.56)
1.75)
P 1
2.0)
Space group
Unit cell
P 32 2 1
P 1 21 1
P 21 21 21
92.1 92.1 164.18
62.9 103.3 76.1
90 93.0 90
59.0 63.0 75.9 48.6 134.3 153.0 90
9
0 90 120
101.1 112.8 94.4
90 90
Total reflections
Unique reflections
Multiplicity
Completeness (%)
Mean I/sigma(I)
Wilson B-factor
R-merge
725065 (34414)
81583 (7832)
8.9 (4.4)
597157 (60933)
133175 (13311)
4.5 (4.6)
539432 (54563)
89118 (9042)
6.1 (6.0)
126530 (12830)
65944 (6644)
1.9 (1.9)
99.59 (96.57)
26.62 (3.32)
23.67
96.18 (95.63)
11.19 (1.22)
14.93
90.73 (92.05)
9.08 (2.02)
95.91 (97.62)
8.23 (2.54)
13.78
23.65
0.04649 (0.3966)
0.04921 (0.451)
0.01592 (0.2106)
0.999 (0.886)
1 (0.969)
0.09883 (1.261)
0.1121 (1.427)
0.05232 (0.6619)
0.998 (0.358)
1 (0.726)
0.1569 (0.7841)
0.1719 (0.8578)
0.06949 (0.345)
0.994 (0.78)
0.999 (0.936)
89095 (9038)
0.05735 (0.2516)
0.08111 (0.3558)
0.05735 (0.2516)
0.996 (0.907)
0.999 (0.975)
65903 (6639)
R-meas
R-pim
CC1/2
CC*
Reflections used in
refinement
81572 (7830)
132517 (13152)
Reflections used for
R-free
4183 (452)
6491 (608)
4458 (428)
3335 (326)
R-work
R-free
0.2006 (0.2858)
0.2202 (0.3105)
0.943 (0.876)
0.937 (0.823)
4138
0.2050 (0.3530)
0.2334 (0.3668)
0.960 (0.626)
0.958 (0.620)
8639
0.1849 (0.2669)
0.2133 (0.2986)
0.954 (0.867)
0.940 (0.834)
8749
0.1864 (0.2446)
0.2216 (0.3016)
0.957 (0.905)
0.939 (0.820)
8306
CC(work)
CC(free)
Number of non-
hydrogen atoms
macromolecules
ligands
3608
7264
112
7264
246
7264
180
solvent
530
480
1263
968
1239
968
862
Protein residues
RMS(bonds)
RMS(angles)
968
0.009
1.00
0.003
0.58
0.004
0.69
0.004
0.61
97.37
Ramachandran
favored (%)
97.46
98.00
97.69
Ramachandran
allowed (%)
2.54
2.00
2.31
2.63
2
5

Ramachandran
outliers (%)
0.00
0.26
0.00
0.76
0.00
0.51
0
Rotamer outliers
0.25
(
%)
Clashscore
Average B-factor
macromolecules
ligands
3.35
30.15
28.81
3.92
22.83
20.95
27.65
33.22
50
2.18
19.00
17.24
21.05
28.88
76
1.85
29.55
28.84
29.75
35.49
46
solvent
39.27
28
Number of TLS
groups
2
6

Figure 1.
Figure 2.
2
7

Figure 3.
2
8

Figure 4.
2
9

Figure 5.
3
0