Comparing naturally occurring glycosylated forms of proline rich antibacterial peptide, Drosocin

Article abstract of DOI:10.1007/s10719-017-9781-8

Antimicrobial peptides (AMPs) are key players of innate immunity. Amongst various classes of AMPs, proline rich AMPs from insects enjoy special attention with few members of this class bearing O-glycosylation as post-translational modification. Drosocin,

Full text of DOI:10.1007/s10719-017-9781-8

Glycoconj J
DOI 10.1007/s10719-017-9781-8
ORIGINAL ARTICLE
Comparing naturally occurring glycosylated forms of proline rich
antibacterial peptide, Drosocin
Deepti S. Lele1 & Gagandeep Kaur1 & Menithalaxmi Thiruvikraman1
&
Kanwal J. Kaur1
Received: 20 February 2017 /Revised: 9 June 2017 /Accepted: 13 June 2017
#
Springer Science+Business Media, LLC 2017
Abstract Antimicrobial peptides (AMPs) are key players of
innate immunity. Amongst various classes of AMPs, proline
rich AMPs from insects enjoy special attention with few mem-
bers of this class bearing O-glycosylation as post-translational
modification. Drosocin, a 19 amino acid glycosylated AMP is
a member of proline rich class, synthesized in the
haemolymph of Drosophila melanogaster upon bacterial
challenge. We report herein the chemical synthesis of drosocin
carrying disaccharide (β-Gal(1 → 3)α-GalNAc) and compar-
ison of its structural and functional properties with another
naturally occurring monoglycosylated form of drosocin i.e.
α-GalNAc-drosocin as well as with non-glycosylated
drosocin. The disaccharide containing drosocin exhibited low-
er potency compared to monoglycosylated drosocin against
all the tested Gram negative bacteria, suggesting the role of
the distal sugar or increase in the sugar chain length on the
activity. Circular dichroism studies failed to demonstrate the
differential effect of sugars on the overall peptide conforma-
tion. Haemolytic and cytotoxic properties of drosocin were
not altered due to an increase in the sugar chain length. In
addition, we have also evaluated the effect of differentially
glycosylated drosocins on two pro-inflammatory cytokines
secreted by murine macrophages or LPS stimulated macro-
phages. All the drosocin forms tested, neither could stimulate
the secretion of TNF-α and IL-6 nor could modulate LPS-
induced levels of TNF-α and IL-6 in murine macrophages.
This study provides insights about naturally occurring two
different glycosylated forms of drosocin.
.
.
.
Keywords Antimicrobial peptides Proline rich Drosocin
.
T-antigen Immunomodulation
Introduction
The universal presence of invading microorganisms com-
mands the need for continuous protection of multicellular or-
ganisms. The innate immunity takes care of the initial interac-
tions between the host and the microbes. It does not depend on
exact antigen recognition, but instead recognizes common
structural characteristics. Antimicrobial peptides (AMPs) are
one such inducible effector molecules of innate immune
mechanism [1]. AMPs are the most important elements of
insects’ defense against harmful bacteria and fungi. The
insect-derived proline rich AMPs include apidaecin [2], a
non-glycosylated AMP and O-glycosylated AMPs like
drosocin [3], pyrrhocoricin [4] and formaecins [5], carrying
a carbohydrate moiety at a conserved threonine residue [6].
Detailed understanding of carbohydrate protein/peptide
crosstalk is difficult because of natural availability of
glycoproteins/glycopeptides in low concentration, only and
inherent heterogeneity of glycosylation. Proline rich antibac-
terial glycopeptides turn out to be an attractive model for
studying the role of glycosylation in peptides’ function.
Earlier we have reported the effect of different monosaccha-
rides, disaccharides and their interglycosidic linkages on the
various properties of two proline rich AMPs, drosocin and
formaecin [7, 8]. A number of glycoproteins have also been
studied to elucidate the effect of the sugar chain length on their
Deepti S. Lele and Gagandeep Kaur contributed equally to this work.
Electronic supplementary material The online version of this article
(doi:10.1007/s10719-017-9781-8) contains supplementary material,
which is available to authorized users.
* Kanwal J. Kaur
kanwal@nii.ac.in
1
National Institute of Immunology, Aruna Asaf Ali Marg, New
Delhi 110067, India

Glycoconj J
structure and function [9]. For example, in case of family 1
carbohydrate binding module (CBM) increase in glycan
length increases the thermolysin resistance of CBM con-
ferring various beneficial properties to the enzyme
targeting cellulose for biomass degradation [10].
Contrary to this, in case of glycosylated antifreeze pep-
tides and glycoproteins (AFGPs), the distal sugar was
not essential for the activity. AFGPs with monosaccha-
ride (αGalNAc-) can display thermal hysteresis activity
[11]. Effect of the increase in glycan side chain or de-
gree of glycosylation on protein stability has also been
established [12].
Materials and methods
Synthesis of glycosylated threonine carrying
Ac4Galβ1 → 3Ac2GalNAcα1 (scheme 1)
Nα-fluoren-9-ylmethoxycarbony-O-(2-azido-2-deoxy-α-
D-galactopyranosyl)-L-threonine benzyl ester (2)
Crude anomeric mixture of Nα-fluoren-9-ylmethoxycarbonyl-
O-(2-azido-2-deoxy-3,4,6-tri-O-acetyl-α-D-galactopyranosyl)-
L-threonine benzyl ester 1 (1.8 g) [3] was dissolved in dry
CH3OH (115 ml). After addition of sodium methoxide (41 ml,
100 mM solution in dry CH3OH), the reaction mixture was
stirred for 2 h at 0 °C. The progress of reaction was monitored
by TLC. After neutralization with ion exchange resin (Amberlite
IR50), the reaction mixture was filtered and concentrated. The
crude product was purified by flash chromatography using
CH2Cl2 as solvent A and 5% CH3OH-CH2Cl2 as solvent B
Drosocin, a 19 amino-acid long peptide is active against
Gram negative bacteria, carrying either a monosaccharide (α-
GalNAc) or a disaccharide (β-Gal(1 → 3)α-GalNAc) on the
threonine residue. Both these glycosylated forms of drosocin
were found to be secreted by Drosophila on bacterial chal-
lenge [3]. Importance of glycosylation on its antibacterial ac-
tivity has been previously discussed. Bulet and co-workers
[13] showed the antibacterial activity pattern of disaccharide
containing drosocin and its non-glycosylated form against
multiple strains of bacteria but comparison of activity of dif-
ferently glycosylated drosocins bearing monosaccharide
and disaccharide with non-glycosylated drosocin was
only studied against E. coli D22. For detailed compara-
tive analysis of antibacterial activity of naturally occur-
ring glycosylated forms of drosocin against various
strains of E. coli, Salmonella and Klebsiella, we have
synthesized drosocins carrying monosaccharide and di-
saccharide in the laboratory. Their antibacterial activities
were also compared with that of non-glycosylated
drosocin. Secondary structures of the peptides were
characterized by circular dichroism (CD) spectrometry.
Cytotoxic effects of monosaccharide and disaccharide
containing drosocins and non-glycosylated drosocin on
mammalian cells were analyzed by haemolytic and MTT
assays. Various antimicrobial peptides are known to reg-
ulate innate and adaptive immune responses in the host.
Cathelicidins like LL-37, are known to act as effector
molecules of the innate immune system and control the
release of pro-inflammatory mediators like TNF-α from
LPS-activated neutrophils and macrophages [14, 15]. It
has also been reported that apidaecin 1b, a non-glycosylated
proline rich AMP, differentially immunomodulates human
macrophages, monocytes and dendritic cells [16]. Hence, we
have analyzed the immunomodulatory effects of differ-
ent glycoforms of drosocin and its non-glycosylated
form on the pro-inflammatory markers, TNF-α and IL-
6, in activated or un-activated murine macrophages.
Comparative analysis of various properties of monosac-
charide and disaccharide containing drosocins will help
in understanding the effect of different sugars in glyco-
sylated variants of the peptide.
1
(gradient of 0–50% B over 40 min) to afford 2 (700 mg). H
NMR (300 MHz, CDCl3): δ 7.74 (d, J = 7.5 Hz, 2H, Ar), 7.59
(dd, J = 2.1 Hz, 7.1 Hz, 2H, Ar), 7.41–7.28 (m, 9H, Ar), 5.90 (d,
J = 9.4 Hz, 1H, NH), 5.29–5.13 (m, 2H, CH2-Ph), 4.88 (d,
J = 3.6 Hz, 1H, H-1), 4.47–4.29 (m, 4H, β-H, α-H, CH2
Fmoc), 4.24–4.19 (m, 1H, CH Fmoc), 4.07 (d, J = 2.4 Hz, 1H,
H-4), 3.95 (dd, J = 2.9 Hz, 10.5 Hz, 1H, H-3), 3.84–3.77 (m, 3H,
H-5, H-6ab), 3.47 (dd, J = 3.4 Hz, 10.1 Hz, 1H, H-2), 1.28 (d,
J = 6.3 Hz, 3H, CH3Thr); 13C NMR (75 MHz, CDCl3): δ 170.6,
157.0 (COFmoc), 144.1, 143.8, 141.5, 135.4, 135.1, 128.9,
128.7, 127.9, 125.4, 125.3, 124.9, 120.2, 99.4 (C-1), 76.4,
70.1, 69.9, 68.3, 68.0, 67.6, 63.1, 60.8, 59.0, 47.3, 18.8 (CH3
Thr). HRMS: calculated for C32H34N4O9Na [M + Na]+
641.2327; found, 641.2609. All data are in agreement with liter-
ature report [17].
Nα-fluoren-9-ylmethoxycarbony-O-(2-azido-4,6-
O-benzylidene-2-deoxy-α-D-galactopyranosyl)-
L-threonine benzyl ester (3)
To a solution of 2 (524 mg, 0.85 mmol)) in CH3CN (4 ml) was
added α, α-dimethoxy-toluene (277 μl, 1.84 mmol), followed by
p-toluenesulfonic acid monohydrate (40 mg, 0.21 mmol) at 0 °C.
The reaction mixture was allowed to stir for 1 h at 0 °C and 3 h at
room temp. The reaction mixture was neutralized using
triethylamine, diluted with CH2Cl2 and washed thoroughly with
water and dried over Na2SO4. The crude residue was purified
using flash chromatography using a gradient of 0–60% EtOAc-
Hexane over 40 min to give the titled compound 3 (395 mg,
1
66%) as a colourless solid. H NMR (300 MHz, CDCl3): δ
7.77 (d, J = 7.4 Hz, 2H, Ar), 7.62 (d, J = 7.4 Hz, 2H, Ar),
7.49–7.46 (m, 2H, Ar), 7.43–7.27 (m, 12H, Ar), 5.77 (d,
J = 9.3 Hz, 1H, NH), 5.56 (s, 1H, CHPh), 5.26 (d, J = 17.6,
2H, CH2-Ph), 4.94 (d, J = 3.3 Hz, 1H, H-1), 4.50–4.42 (m, 3H,
βHThr, CH2Fmoc), 4.33 (dd, J = 10.3, 7.4 Hz, 1H, H-3), 4.26–

Glycoconj J
4.22 (m, 3H, H-6a, αHThr, CHFmoc), 4.12–4.02 (m, 3H, H-6b,
H-5, H-4), 3.70 (br s, 1H, OH), 3.53 (dd, J = 10.5, 3.5 Hz, 1H,
H-2), 1.29 (d, J = 6.4 Hz, 3H, CH3Thr). 13C NMR (75 MHz,
CDCl3): δ 170.3, 157.0 (COFmoc), 144.1, 143.9, 141.5, 137.4,
135.2, 129.6, 128.9, 128.8, 128.6, 127.9, 127.3, 126.4, 125.4,
120.2, 101.5, 99.4 (C-1), 76.5, 75.5, 69.3, 67.9, 67.7, 67.6, 63.5,
61.4, 58.9 53.6, 47.4 (CHFmoc), 18.9 (CH3Thr). HRMS: calcu-
lated for C39H39N4O9 [M + H]+ 707.2718; found, 707.4371. All
data are in agreement with literature report [17].
completion of the reaction, the mixture was concentrated and
dried to afford compound 5. To the solution of 5 (360 mg) in
pyridine (5 ml) was added acetic anhydride (2.5 ml) at 0 °C.
The reaction was allowed to warm and stirred for 6 h at room
temperature. The reaction mixture was concentrated and puri-
fied by flash chromatography using gradient of 0–35%
EtOAc-Hexane over 30 min, to yield pure compound 6
1
(270 mg, 67.5%). H NMR (300 MHz, CDCl3): δ 7.78 (d,
J = 7.5 Hz, 2H, Ar), 7.62 (d, J = 7.3 Hz, 2H, Ar), 7.44–7.29
(m, 9H, Ar), 5.67 (d, J = 9.4 Hz, 1H, NH), 5.46 (d, 1H,
J = 3.1 Hz, H-4′), 5.37 (d, 1H, J = 3.3 Hz, H-4), 5.29–5.16
(m, 3H, CH2-Ph, H-2′), 5.01 (dd, J = 10.5, 3.3 Hz, 1H, H-3′),
4.84 (d, J = 3.7 Hz, 1H, H-1), 4.70 (d, J = 7.7 Hz, 1H, H-1′),
4.54–4.40 (m, 3H, CH2Fmoc, βH Thr), 4.37–4.31 (m, 1H,
αHThr), 4.16–4.07 (m, 4H, CHFmoc, H-3, H-6a’b’), 4.04–
3.89 (m, 4H, H-5′, H-5, H-6ab), 3.53 (dd, J = 10.7, 3.7 Hz,
1H, H-2), 2.15, 2.13, 2.06, 2.04, 2.03, 1.99 (6 s, 3H each,
6XOAc), 1.32 (d, J = 6.4 Hz, 3H, CH3-Thr). 13C NMR
(75 MHz, CDCl3): δ 170.6, 170.4, 170.3, 170.2, 170.1,
169.7, 156.9 (CO Fmoc), 144.0, 143.8, 141.5, 135.1, 129.1,
128.9, 128.8, 128.7128.0, 127.3, 127.2, 125.3, 125.2, 120.2,
101.7 (C-1′), 99.5 (C-1), 75.0, 71.0 (C-3′), 69.5, 69.0 (C-4′),
68.1, 67.9, 67.6, 67.0 (C-4), 63.1, 61.2, 60.5, 59.9 (C-2), 58.9,
47.3, 21.2, 20.9, 20.8, 20.7, 18.7 (CH3Thr). HRMS calculated
for C50H56N4O20Na 1055.3488; found 1055.327.
Nα-fluoren-9-ylmethoxycarbony-O-[2-azido-4,6-
O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-
D-galactopyranosyl)-α-D-galactopyranosyl]-L-threonine
benzyl ester (4)
A reaction mixture of 3 (510 mg, 0.72 mmol), per-O-acetylated
trichloroacetimidate derivative of Galactose (711 mg, 1.44 mmol)
[18] and activated 4A° molecular sieves (600 mg) in dry CH2Cl2
(10 ml) was stirred for 30 min. at room temperature under argon.
TMSOTf (26 μl, 0.14 mmol) was added to the reaction mixture
at 0 °C. The reaction mixture was warmed to room temperature
and stirred until completion. The reaction was quenched with a
few drops of triethylamine. The reaction mixture was diluted
with CH2Cl2 and filtered through Celite. The filtrate was succes-
sively washed with satd NaHCO3, H2O and dried over MgSO4
and concentrated. Purification by flash chromatography using
gradient of 0–40% EtOAc-Hexane over 40 min. Afforded 4
Nα-fluoren-9-ylmethoxycarbony-O-[2-acetamido-4,
6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-
D-galactopyranosyl)-α-D-galactopyranosyl]-L-threonine
benzyl ester (7)
1
(500 mg, 66.8%). H NMR (300 MHz, CDCl3): δ 7.78 (d,
J = 7.5 Hz, 2H, Ar), 7.62 (d, J = 7.4 Hz, 2H, Ar), 7.55–7.52
(m, 2H, Ar), 7.44–7.29 (m, 12H, Ar), 5.78 (d, J = 9.4 Hz, 1H,
NH), 5.54 (s, 1H, PhCH(O)2), 5.41 (d, J = 3.3 Hz, 1H, H-4′),
5.34–5.22 (m, 3H, H-2′, CH2-Ph), 5.04 (dd, J = 10.4, 3.4 Hz, 1H,
H-3′), 4.94 (d, J = 3.5 Hz, 1H, H-1), 4.79 (d, J = 7.9 Hz, 1H,
H-1′), 4.56–4.45 (m, 2H, CH2Fmoc), 4.36–4.32 (m, 1H, H-6a),
4.30–4.19 (m, 4H, βHThr, αHThr, CH Fmoc, H-6a’), 4.16–4.08
(m, 2H, H-3, H-4), 4.04–3.91 (3H, H-6b’, H-6b, H-5′), 3.75 (dd,
J = 10.8, 3.6 Hz, 1H, H-2), 3.65 (br s, 1H, H-5), 2.17, 2.05, 2.03,
1.99 (4 s, 3H each, 4XOAc), 1.31 (d, J = 6.3 Hz, 3H, CH3Thr).
13C NMR (75 MHz, CDCl3): δ 170.5, 170.4, 170.3, 169.6, 156.9
(CO Fmoc), 144.1, 143.8, 141.5, 137.7, 135.1, 129.2, 128.9,
128.8, 128.7128.4, 127.9, 127.3, 127.2, 126.3, 125.4, 125.3,
120.2, 102.6, 100.9 (C-1′), 99.6 (C-1), 76.3, 76.1, 75.8, 71.2,
71.1, 69.2, 68.8, 67.9, 67.5, 67.1, 63.7, 61.6, 59.3, 58.9, 47.3
(CH Fmoc), 20.9, 20.8, 19.0 (CH3Thr). HRMS calculated for
C53H56N4O18Na 1059.3590; found 1059.3727.
To a solution of compound 6 (210 mg, 0.20 mmol) in pyridine
(7 ml), thiolacetic acid (7 ml) was added drop-wise at 0 °C.
The reaction mixture was allowed to attain room temperature
and stirred for overnight. The reaction mixture was diluted
with toluene (15 ml) and the solvents were evaporated. The
residue was co-concentrated thrice from toluene. Crude prod-
uct was purified using flash chromatography following gradi-
ent of 0–60% EtOAc-Hexane over 40 min, which yielded 7
1
(170 mg, 80%) as a white amorphous solid. H NMR
(300 MHz, CDCl3): δ 7.79 (d, J = 7.3 Hz, 2H, Ar), 7.62 (d,
J = 7.0 Hz, 2H, Ar), 7.45–7.30 (m, 9H, Ar), 5.77 (d,
J = 8.8 Hz, 1H, NH), 5.53 (d, J = 9.4 Hz, 1H, NH), 5.37 (d,
2H, J = 2.4 Hz, H-4′, H-4), 5.29–5.05 (m, 3H, CH2-Ph, H-2′),
4.96 (dd, J = 10.5, 3.0 Hz, 1H, H-3′), 4.82 (d, J = 2.4 Hz, 1H,
H-1), 4.59–4.40 (m, 5H, H-1′, CH2-Fmoc, βHThr, αHThr),
4.26–4.09 (m, 6H, H-2, H-5, H-5′, H-6a’b’, CHFmoc), 3.99–
3.87 (m, 2H, H-6ab), 3.81 (dd, J = 10.7, 2.2 Hz, 1H, H-3),
2.16, 2.13, 2.07, 2.04, 2.03, 1.98 (6 s, 21H, 7XOAc), 1.29–
1.23 (m, 3H, CH3Thr). 13C NMR (75 MHz, CDCl3): δ 170.9,
170.6, 170.5, 170.3, 170.2, 170.1, 169.8, 156.6 (CO Fmoc),
143.9, 143.8, 141.5, 134.6, 129.2, 129.1, 128.8, 128.7,
128.6128.0, 127.3, 125.1, 120.3, 100.9 (C-1′), 100.3 (C-1),
Nα-fluoren-9-ylmethoxycarbony-O-[2-azido-4,6-di-
O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-
O-acetyl-β-D-galactopyranosyl)-α-D-galactopyranosyl]
-L-threonine benzyl ester (6)
Compound 4 (402 mg, 0.39 mmol) was dissolved in 80% of
acetic acid in water (12 ml) and stirred for 1.5 h at 70 °C. After

Glycoconj J
73.3, 71.0, 70.9 (C-3′), 69.9, 69.0 (C-4′), 68.1, 68.0, 67.3, 67.0
(C-4), 63.1, 61.2, 60.6, 58.9, 48.9, 47.4 (CH Fmoc), 23.5,
21.2, 20.9, 20.8, 20.7, 18.5 (CH3Thr). HRMS: calculated for
C52H61N2O21 [M + H]+ 1049.3767; found, 1049.5974.
of monoglycosylated drosocin (M-drosocin) and non-
glycosylated drosocin (n-drosocin) have been discussed pre-
viously [7].
Antibacterial activities
Nα-fluoren-9-ylmethoxycarbony-O-[2-acetamido-4,
6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-
O-acetyl-β-D-galactopyranosyl)-α-D-galactopyranosyl]
-L-threonine (I)
Minimum inhibitory concentrations (MICs) against various
bacterial strains such as E. coli ATCC 25922, E. coli ATCC
11755, E. coli ATCC 35218, E. coli ATCC 8739, E. coli
ATCC 10536, E. coli ATCC 9637, E. coli ATCC ML35p,
S. typhimurium ATCC 14028, S. typhi Vi+, S. enterica
NCTC 6017, K. pneumoniae ATCC 13883 and
K. pneumoniae ATCC 700603 were determined for peptides
using broth microdilution assay in Mueller Hinton broth.
Bacteria growing in logarithmic growth phase were washed
thrice with Phosphate Buffer (PB 10 mM, pH 7.4) and diluted
in Muller Hinton broth to adjust A600 = 0.001 corresponding
to 3-5x105CFU/ml. Ninety μL of this culture was aliquoted in
96 well plate and 10 μL of varying concentrations of different
peptides were added. The plate was incubated at 30 °C for
22 h. The MIC is defined as lowest concentration of the pep-
tide that inhibits measurable growth of an organism. Each test
condition was set up in duplicate and the experiment was
repeated at least thrice to calculate the average.
To a solution of 7 (660 mg, 0.63 mmol) in CH3OH (10 ml)
was added 10% Pd/C (660 mg). The mixture was stirred under
one atmosphere of hydrogen gas for 2 h. The catalyst was
removed by filtration and after solvent evaporation, the resi-
due was purified by flash chromatography using CH2Cl2 as
solvent A and 10% CH3OH-CH2Cl2 as solvent B (gradient of
0–70% B over 50 min) to afford I (518 mg, 86%) as a white
1
solid. H NMR (300 MHz, CD3OD): δ 7.82 (d, J = 7.3 Hz,
2H, Ar), 7.69 (d, J = 7.3 Hz, 2H, Ar), 7.43–7.31 (m, 4H, Ar),
5.37–5.34 (m, 2H, H-4′, H-4), 4.99–4.97 (m, 2H, H-3′, H-2′),
4.63–4.49 (m, 4H, H-1, H-1′, CH2Fmoc), 4.38–4.26 (m, 3H,
βHThr, H-2, CHFmoc), 4.22–4.07 (m, 5H, αHThr, H-6b,
H-6b’, H-5, H-5′), 3.99–3.93 (m, 2H, H-6ª, H-6ª’), 3.87 (d,
J = 11.1 Hz, 3.2 Hz, 1H, H-3), 2.12, 2.09, 2.04, 2.02, 2.00,
1.96, 1.93 (7 s, 21H, 7XOAc), 1.23 (d, J = 6.6 Hz, 3H,
CH3Thr).. 13C NMR (75 MHz, CD3OD): δ 172.1, 171.8,
170.9, 170.7, 170.6, 170.1, 169.6, 157.7 (CO Fmoc), 143.9,
143.8, 141.3, 127.5, 126.8, 124.7, 124.6, 119.7, 119.6 (Ar C),
101.1 (C-1′), 99.5 (C-1), 76.0, 73.6, 70.8, 70.3, 69.8, 68.7,
67.6, 67.1, 66.3, 62.9, 60.9, 58.5, 53.4, 21.8, 19.4, 19.3,
19.2, 19.1, 17.8 (CH3Thr). HRMS: calcd for C45H55N2O21
[M + H]+ 959.3297; found, 959.5693.
Killing kinetics
The kinetics of antibacterial activity against E. coli ATCC
25922 by non-glycosylated and glycosylated forms of
drosocin was performed in Mueller Hinton media, 10 mM
Phosphate buffer containing 150 mM NaCl, pH 7.4 (PBS)
and Dulbecco’s Modified Eagle’s Medium (DMEM). Mid-
log phase bacteria (approximately 3-4x105CFU/ml) were in-
cubated at 30 °C up to 6 h or 12 h without (only cells) and with
peptide. Aliquots were removed at different time intervals,
diluted in PB and plated in Mueller-Hinton solid medium to
allow colony counts. All assays were repeated at least once,
with the difference between assays being ≤1 log10 CFU/ml.
The results presented in Figs. 1, S1 and S2 were from a single
representative experiment.
Peptide synthesis
Disaccharide containing drosocin [GKPRPYSPRPT(β-
Gal(1 → 3)αGalNAc) SHPRPIRV; Di-drosocin] used in this
study was chemically synthesized by solid-phase method on
an automated peptide synthesizer (433A; Applied
Biosystems, Foster City, CA, USA), employing Fmoc-meth-
odology. For synthesizing glycosylated peptide, pre-
synthesized glycosylated amino acid was added and coupled
manually on the growing polypeptide chain. The peptide was
cleaved from the peptide-resin by treatment with a cleavage
mixture containing phenol (0.75 g), EDT (0.25 ml), TFA
(10 ml), thioanisole (0.5 ml) and water (0.5 ml). The crude
peptide was purified by semi-preparative HPLC (Waters,
USA) using C18 column (Waters XBRIDGE™ BEH 130
reverse phase C18 column, 19 X 250 mm, 10 μm, spherical)
as reported previously [8]. The purified glycosylated peptide
was de-acetylated using aqueous 5% hydrazine hydrate and
then purified again using HPLC. The peptide was character-
ized using mass spectrometry. Synthesis and characterization
Circular-dichroism spectroscopy
The circular-dichroism (CD) spectra of all peptides (50 μM)
were obtained at 25 °C in a 1 mm path-length cuvette in the
range of 250–190 nm using CD spectrometer (J-815 JASCO
Corporation, Japan). Five scans with a speed of 100 nm/min
were averaged and analyzed using JASCO spectra analysis
software. The spectra were recorded in 10 mM PB pH 7.4,
90% TFE in water and 10 mM SDS in 10 mM PB pH 7.4.
Results were expressed as mean residue ellipticity in [θ] (Deg-
cm2/dmol).

Glycoconj J
Haemolytic assay
concentrations of pro-inflammatory cytokines TNF-α and
IL-6 in the supernatants of stimulated RAW 264.7 cells by
sandwich ELISA using commercially available ELISA Kits
(BD Biosciences, San Diego, USA) in accordance with the
manufacturer’s description. Cells were seeded in 24-well tis-
sue culture plates at a density of 5 × 105 cells/ml and incubated
overnight at 37 °C in 5% CO2. Cells were then incubated at
least in triplicate for 24 h in the presence of medium alone or
with E. coli O111:B4 LPS (20 ng/ml) (Sigma Chemical Co.,
St Louis, USA), different concentrations (6.25 μM, 12.5 μM,
25 μM, 50 μM and 200 μM) of synthesized peptides or a
combination of LPS and peptide or polymyxin B.
Polymyxin B (7.2 μM = 10 μg/ml) was used as a positive
control. Supernatants were collected and stored at −20 °C until
use. Each experiment was set up in triplicate and the results
have been averaged from three independent experiments.
Haemolytic assay was done as described previously [7]. In brief,
a 200 μl aliquot of packed rat erythrocytes volume was washed
with cold PBS (10 mM PB, pH 7.4, 150 mM NaCl) at 4 °C till
the supernatant was colourless, which was discarded each time.
The pellet was resuspended in PBS, diluting the cells to 2% of
their packed volume. The erythrocytes (90 μl) were plated in a
96-well microtiter plate (Greiner Bio-one, Wemmel, Belgium)
along with 10 μl of peptide solution corresponding to final con-
centration of 200 μM added to the wells and the plate was incu-
bated at 37 °C for 90 min. Any haemolytic activity would man-
ifest as rupture of RBCs and release of haemoglobin into the
solution. Subsequently, the plate was centrifuged at 4000 rpm
for 10 min at 4 °C to pellet intact cells and 75 μl of supernatant
was transferred to a flat bottom microtiter plate (Greiner Bio-one,
Wemmel, Belgium). Its absorbance was read at 405 nm and the
percentage of RBCs lysis was calculated. The haemolysis affect-
ed by only PBS and 1% TritonX-100 were considered as nega-
tive control and 100% haemolysis, respectively. Each test condi-
tion was set up in triplicate and the results have been averaged
from three independent experiments.
Statistical analysis
Statistical analysis of variance was performed using OriginPro
8.5 and significance of differences (p ≤ 0.01) with respect to
control values were calculated by paired t tests.
MTT assay
Results and discussion
Cytotoxicity of the synthetic peptides was determined by stan-
dard MTT assay following the protocol as previously described
[8]. The method is based on the reduction of the salt,
Methylthiazolyldiphenyl Tetrazolium Bromide (MTT) into a
crystalline blue formazan product by the cellular oxidoreductases
of viable cells. The synthesized peptides were incubated with
murine macrophages cell lines, J774 and RAW 264.7
(2.5 × 104 cells/well) grown in DMEM. Following 24 h incuba-
tion with peptides (200 μM), cells were washed and then incu-
bated with fresh culture media and 100 μl MTT (Sigma,
0.25 mg/ml) in 0.1 M PBS, pH 7.4 at 37 °C in a humid atmo-
sphere with 5% CO2 for 3 h. Media was then gently aspirated
from test cultures and 100 μl of Dimethyl Sulphoxide (DMSO)
was added to the wells. The plates were shaken for 2 min before
taking the absorbance at 570/630 nm in a microtiter plate reader.
The percentage of viability was calculated as AT/AC × 100;
where AT and AC are the absorbances of treated and negative
control cells, respectively. In the positive and negative controls,
the peptide solution was substituted by the same volumes of
ethanol and PBS, respectively. Each test condition was set up
in triplicate and the results have been averaged from three inde-
pendent experiments.
Synthesis of drosocin carrying disaccharide
Two glycosylated forms of drosocin carrying monosaccharide
(α-GalNAc) and disaccharide (Galβ1 → 3GalNAcα) were
found to be secreted by Drosophila upon bacterial challenge
[3]. In this report, we have compared the effect of these two
sugars on the antibacterial potential of drosocin. The comparative
study of other properties like haemolytic, cytotoxic and
immunolmodulatory activities of these two isoforms and non-
glycosylated form was also undertaken. Monosaccharide, Tn an-
tigen containing drosocin (M-drosocin) and non-glycosylated
drosocin (n-drosocin) were synthesized in the laboratory previ-
ously [7]. To synthesize drosocin carrying disaccharide, first the
critical building block BNα-fluoren-9-ylmethoxycarbony-O-[2-
acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-
acetyl-β-D-galactopyranosyl)-α-D-galactopyranosyl]-L-
threonine^ (I) (Scheme 1) was prepared and then incorporated
onto the growing polypeptide chain as discussed earlier [7] using
solid phase peptide synthesis. Briefly, the peptide chain was first
assembled on the resin by standard Fmoc/DCC-HOBt protocol
until the glycosylated threonine residue position. The glycosylat-
ed threonine I was then coupled to the NH2-peptide resin by
Fmoc/HBTU protocol [19]. Further elongation of the peptide
chain was carried out according to the standard Fmoc/DCC-
HOBt protocol. The deprotection and cleavage of peptide from
resin provided the glycopeptide with acetyl groups protected
sugar. The purified glycosylated peptide was deacetylated and
Effects of peptides on the secretion of TNF-α and IL-6
in murine macrophages (RAW 264.7)
The immunomodulatory effects of drosocin peptides on mu-
rine macrophages, were assessed by measuring the

Glycoconj J
Scheme 1 Synthetic scheme for synthesis of Nα-fluoren-9-
ylmethoxycarbony-O-[2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-α-D-galactopyranosyl]-
L-threonine(I) from compound 1 (Nα-fluoren-9-ylmethoxycarbonyl-O-
(2-azido-2-deoxy-3,4,6-tri-O-acetyl-α-D-galactopyranosyl)-L-threonine
benzyl ester)
finally, the disaccharide containing drosocin (Di-drosocin) was
repurified using HPLC and characterized using MALDI-TOF
(Table 1 and see supplementary data).
Salmonella and some of the E. coli strains. Di-drosocin and n-
drosocin showed the similar antibacterial activity against both of
the Klebsiella strains and E. coli ATCC 11775, E. coli ATCC
8739 and E. coli ML35p. M-drosocin exhibited the lowest MIC
value compared to that of Di-drosocin and n-drosocin against all
the tested Gram negative bacterial strains of E. coli, Salmonella
and Klebsiella (Table 2). Thus, aforementioned results indicated
that glycosylation of the peptide as well as increase in sugar chain
length affected the antibacterial activity of the peptide. In the
earlier studies Bulet et al. [13] reported that monosaccharide
(α-GalNAc) containing drosocin possessed lower antibacterial
activity in comparison with that of drosocin bearing disaccharide
against E. coli D22. Contrary to it, our studies showed that M-
drosocin was more active than Di-drosocin against all the tested
Gram negative bacterial strains. These differences in the results
may arise because of the difference in the bacterial strain as it has
been shown that magnitude of antibacterial activity of peptide
varies in different strains.
Antibacterial activity
The antibacterial activity of disaccharide containing drosocin
[GKPRPYSPRPT(βGal(1 → 3)αGalNAc)SHPRPIRV; Di-
drosocin] was compared with monosaccharide carrying drosocin
[GKPRPYSPRPT(αGalNAc)SHPRPIRV; M-drosocin], and
non-glycosylated drosocin (GKPRPYSPRPTSHPRPIRV; n-
drosocin), using microbroth dilution assay (Table 2). It was ob-
served that disaccharide containing drosocin displayed two fold
higher MIC against E. coli ATCC 25922, E. coli ATCC 9637 and
S. typhimurium ATCC14028 whereas four fold higher MIC
against E. coli ATCC 35218, E. coli ATCC 10536, S. typhi
Vi + and S. enterica NCTC 6017 in comparison with that of
M-drosocin. Moreover, Di-drosocin exhibited eight fold higher
MIC against E. coli ATCC 11775, E. coli ATCC 8739, E. coli
ML-35p and K. pneumoniae ATCC 13883 and sixteen fold
higher MIC against K. pneumonia ATCC 700603 in comparison
with that of M-drosocin. However, Di-drosocin resulted around
two to four times lower MIC than that of n-drosocin against
Time killing assay
The time-killing experiments were carried out in Muller
Hinton (MH) media to follow kinetics of inactivation of

Glycoconj J
Table 1 Amino acid sequences and mass characterization of different forms of drosocin
Peptide
Name
Sequence
MALDI-TOF MALDI-TOF
MS MS
[M+H]Calcd* [M+H]Exptl*
n-drosocin
2198.2436
2198.4417
GKPRPYSPRPTSHPRPIRV
M-drosocin
2401.3230
2401.3713
HO
OH
O
HO
AcHN
O
GKPRPYSPRPTSHPRPIRV
2563.3757
2563.3860
HO
OH
HO
OH
O
O
Di-drosocin
O
HO
AcHN
OH
O
GKPRPYSPRPTSHPRPIRV
*Calcd and Exptl are abbreviations used for calculated and experimental values, respectively
E.coli ATCC 25922 by different glycosylated forms of
drosocin and non-glycosylated drosocin and to establish
whether these peptides are bacteriostatic or bactericidal. A
time course analysis of the inhibitory activity showed that
M-drosocin was the most active peptide, eliminating E. coli
at MIC over a period of 120 min. At MIC value, the number of
viable bacteria decreased tenfold within 10 min by M-
drosocin and within 80 min by Di-drosocin. At 2 fold MIC,
M-drosocin and Di-drosocin required 40 and 120 min, respec-
tively, to completely kill the bacteria. The non-glycosylated
drosocin acted more slowly. It took 120 min to reduce the
viable cells counts to 50% at its MIC value and even at 2 fold
MIC, non-glycosylated drosocin failed to totally eliminate
E. coli bacteria up to 360 min of treatment. The results showed
Table 2 Minimum inhibitory
concentrations (in μM) of
Bacterial strain
n-drosocin
M-drosocin
Di-drosocin
different glycosylated forms of
E. coli ATCC 25922
E. coli ATCC 11775
E. coli ATCC 35218
E. coli ATCC 8739
4
2
1
2
2
drosocin and non-glycosylated
drosocin against Gram negative
bacteria
0.25
0.25
0.25
0.5
2
2
1
2
2
E. coli ATCC 10536
E. coli ATCC 9637
4
2
6
4
E. coli ML35p
2
0.25
0.5
0.25
0.5
0.5
1
2
S. typhimurium ATCC 14028
S. typhi Vi+
4
1
4
1
S. enterica NCTC 6017
K. pneumoniae ATCC 13883
K. pneumoniae ATCC 700603
4
2
4
4
16
16

Glycoconj J
that all the three peptides were bactericidal and M-drosocin
had lower MIC and faster bactericidal activity in comparison
to that of Di-drosocin and n-drosocin (Fig. 1).
In our earlier studies [8] we have shown the correlation
between the antibacterial activity of the glycopeptides and
their internalization into the bacterial cells. Antibacterial pep-
tide possessing lower MIC value was internalized faster in
comparison to the antibacterial peptide having higher MIC
value. The differences observed in the antibacterial activity
of different forms of drosocin may be due to differences in
their rate of internalization in the bacterial cells. It has been
reported that small changes in carbohydrate composition are
sufficient to induce conformational changes in the peptide
[20]; hence, the variation in the antibacterial activity of M-
drosocin and Di-drosocin can also be attributed to the dissim-
ilarities in their conformation and resulting differences in their
binding affinity for target molecules [21, 22]. Thus, size of the
sugar may play a key role in modulating the biological activity
of glycopeptide.
Effect of sugar chain length on antimicrobial activity of
drosocin corroborates the earlier observations where stimula-
tory ability of T-helper cell epitope was found to be hampered
with extended sugar chain [23]. On the contrary, for CLV3
glycopeptide, its biological activity increased progressively
as the sugar chain length increased [24]. Previous studies on
glycosylated enkephalin based opioid analgesics peptides,
proved that peptides with disaccharide exhibited better perme-
ability across the blood brain barrier and showed increased
potency when administered intravenously [25]. Whereas stud-
ies on modified IgG molecules showed that even a monosac-
charide residue at the glycosylation site was sufficient to ex-
hibit IgG subclass activity in the absence of further extended
glycosylation and branching [26].
Circular dichroism spectroscopy
The comparative circular dichroism (CD) spectral analysis
was carried out to measure the conformational effects of
monosaccharide and disaccharide on the drosocin backbone
in different solvents like PB (10 mM, pH 7.4), 90% TFE/
water, and 10 mM SDS (Fig. 2). The CD spectra of glycosyl-
ated and non-glycosylated forms of drosocin in PB are shown
in Fig. 2a. The overall spectral profiles for all the peptides
were similar whereas the absolute values of the mean residue
ellipticities varied among the peptides. The CD spectra of all
the different forms of drosocin displayed random coil second-
ary structure in PB, with a strong negative band at around
200 nm. In 90% TFE/water and 10 mM SDS, both the glyco-
sylated forms and non-glycosylated peptide exhibited spectral
alterations as shown by the deviation in the curves to a more
positive ellipticity (Fig. 2b and c). All the peptides were un-
able to acquire the properly ordered secondary structure even
in 90% TFE/water as well as 10 mM SDS. The CD spectra of
both glycopeptides indicated absence of any regular second-
ary structure and displayed similar spectral characteristics and
were found to be similar to CD spectra of non-glycosylated
drosocin.
Fig. 1 Killing kinetics of different forms of drosocin against E. coli
ATCC 25922 in MHB. Kinetics and dose response effect of the
bactericidal activity of (a) n-drosocin, (b) M-drosocin and (c) Di-
drosocin on E. coli at their 1 X MIC and 2 X MIC values. M-drosocin
exhibits faster bactericidal activity in comparison with that of Di-drosocin
and n-drosocin
Thus, CD studies of disaccharide containing drosocin
showed that its secondary structure was not affected by in-
crease in sugar chain length as CD spectra of Di-drosocin were
similar to that of M-drosocin in different environments. The

Glycoconj J
Fig. 2 Circular Dichroism spectra of drosocin analogs showed
characteristic random coil secondary structure in different environments
and are unaltered with change in the chain length of sugar. Circular
Dichroism spectra of M-drosocin, Di-drosocin, n-drosocin in (a)
10 mM PB pH 7.4 (b) 90% TFE/water (c) 10 mM SDS. CD spectra of
all three peptides show random coiled conformation, which was observed
to be unaltered in presence of different environments. Increase in sugar
length did not show any conformational variation as observed by CD
structural studies of a mucin fragment from CD43 by NMR
[27] also suggested that the distal sugar residue did not influ-
ence the overall peptide conformation. Our results indicated
that increase in carbohydrate chain length in drosocin can
modulate the antibacterial activity but its effect on conforma-
tion of peptide could not be resolved using CD.
both the glycosylated and non-glycosylated forms of drosocin
displayed the bactericidal activity against E. coli ATCC 25922
in DMEM (Fig. S2). Thus, increasing sugar chain length in Di-
drosocin was observed to be non-toxic up to a concentration of
200 μM to mammalian erythrocytes tested as well as to macro-
phages tested similar to that of M-drosocin and n-drosocin.
Thus, we observed that M-drosocin exhibited higher anti-
bacterial activity compared to Di-drosocin and non-
Haemolytic activity
Haemolytic activity of drosocin carrying disaccharide was
examined on murine erythrocytes and was compared with that
of M-drosocin and n-drosocin. Di-drosocin displayed less
than 10% haemolytic activity at the concentration of
200 μM when tested on 2% (vol/vol) suspensions of rat blood
cells, similar to that of M-drosocin and n-drosocin (Fig. 3). All
the different forms of drosocin were found to be non-toxic to
rat blood cells even at the concentration of 200 μM which is
quite higher than the concentrations at which drosocin forms
displayed bactericidal activity against E. coli ATCC 25922 in
PBS (Fig. S1). Moreover, Di-drosocin behaved similar to that
of the monoglycosylated drosocin and thus, increasing the
length of sugar chain did not affect the haemolytic activity
of drosocin.
Cytotoxic activity
Fig. 3 Haemolytic activity of glycosylated as well as non-glycosylated
forms of drosocin on murine erythrocytes. Percentage haemolysis was
calculated after exposure of murine erythrocytes to 200 μM of n-drosocin,
M-drosocin and Di-drosocin. Erythrocytes incubated with 10 mM PBS alone
served as the negative control and cells lysed using 1% Triton X-100 were
used to measure 100% lysis, which served as a positive control. All the
peptides studied do not show haemolytic activity. The results represent the
mean S.E.M. of at least three independent experiments performed in
triplicate. **: p < 0.01 compared to the PBS control; ‘ns’ means there was
no statistical significance from the PBS control
The cytotoxic potential of the disaccharide containing drosocin
was also compared with that of monoglycosylated and non-
glycosylated forms of drosocin by following incubation of mu-
rine macrophages cell lines, J774 and RAW 264.7 in DMEM for
24 h with these peptides using MTT assay. All the different
forms of drosocin caused no decrease in cell viability of J774
and RAW 264.7 in comparison with that of control group (no
peptide) (Fig. 4) even at the concentration of 200 μM at which

Glycoconj J
Fig. 4 Cytotoxic activity of drosocins on macrophage cell lines. Percentage
cell viability was calculated for n-drosocin, M-drosocin and Di-drosocin
against murine macrophage cell lines, J774 and RAW 264.7 at
concentrations of 200 μM following standard MTT assay considering cell
viability in presence of only PBS as 100% as depicted by no peptide.
Macrophages showed less than 5% cell viability in presence of 70%
ethanol which served as a positive control. The results represented in the
bar graph depict the mean S.E.M. of at least three independent
experiments performed in triplicate. Two asterisks (**) means statistically
different (p < 0.01) from the no-peptide control while ‘ns’ means there was
no statistical significance from the no-peptide control
glycosylated drosocin. Increased sugar chain length affected
the antibacterial activity of drosocin but neither altered the
random coil conformation of drosocin according to CD spec-
tra nor changed the toxicity against mammalian cells tested.
Effect of peptides on secretion of TNF-α and IL-6
in macrophages
We have observed that all the drosocin forms are non-toxic to
murine macrophages. So, the effect of different concentrations
of these peptides on secretion of pro-inflammatory markers,
TNF-α and IL-6, in murine macrophage cells was investigated
in presence and absence of LPS. None of the drosocin
forms could stimulate macrophages to secrete TNF-α
and IL-6 up to the concentration of 200 μM (Figs S3
and S4). LPS is known to activate the macrophages to
secrete the pro-inflammatory markers. Polymyxin B was
used as a positive control which inhibited LPS-induced
activation of macrophages. The different drosocin forms
did not modulate the LPS induced secretion of TNF-α
and IL-6 in murine macrophages (Fig. 5). Even their
200 μM concentration, at which all the drosocin forms
displayed the bactericidal activity against E. coli ATCC
Fig. 5 Effect of drosocins on LPS stimulated secretion of TNF-α and IL-6 in
macrophages. RAW 264.7 cells were treated with LPS (20 ng/ml) in the
absence or presence of different concentrations of peptides (12.5 μM,
25 μM and 50 μM) and polymyxin B (7.2 μM) for 24 h. Concentrations
of (a) TNF-α and (b) IL-6 in the cell supernatant were measured by ELISA.
Levels of cytokines present in the cell supernatant of macrophages with
medium alone were subtracted from treatments as background cytokine
levels. Polymyxin B served as a positive control which inhibited the LPS
induced secretion of cytokines. Data shown are representative values of three
independent experiments performed in triplicate. n.d. (not detectable)
indicates that cytokine levels were below detection level. **: p < 0.01
compared to the LPS; ‘ns’ means there was no statistical significance from
the LPS

Glycoconj J
25922 in DMEM, could not influence LPS-induced se-
cretion of TNF-α and IL-6 in murine macrophages (Fig.
S4). Thus, increasing the sugar chain length in drosocin
doesn’t affect secretion of TNF-α and IL-6 in LPS ac-
tivated murine macrophages. Apidaecin 1b, a proline
rich antimicrobial peptide from the same class, has been
earlier observed to stimulate the human macrophages
[16]. This peptide was also found to partially inhibit
TNF-α/IL-6 from LPS activated macrophages at subop-
timal concentration [16]. On the contrary, recent report
from Fritsche et al. [28] could not observe any such
immunomodulatory effects of apidaecin 1b and its de-
rivatives on any pro-inflammatory markers produced by
murine macrophages stimulated with or without LPS.
Similarly, our results exhibit the absence of induction
of cytokines, TNF-α and IL-6, and inhibition of LPS-
dependent effects for non-glycosylated and glycosylated
forms of drosocin when studied in murine macrophages. It has
been reported in the literature [29] that cell surface pattern
recognition receptors (PRRs) vary in immune cells of different
species and these PRRs are responsible for the induction of a
pro-inflammatory immune response. Therefore, this might be
one of the reasons for the differences in the results arising due
to use of immune cells from different sources. Absence of
immunomodulation or specifically pro-inflammatory re-
sponses has also been observed in other AMPs [30].
drosocin and Di-drosocin or n-drosocin could exhibit the bac-
tericidal effect at 50 μM and 200 μM, respectively. Di-
drosocin did not modulate the expression of pro-
inflammatory markers, TNF-α and IL-6 in the tested murine
macrophages in presence and absence of LPS in vitro similar
to that of M-drosocin and n-drosocin. This study provides a
systematic comparison of differently glycosylated forms of
drosocin and insights for understanding the effect of sugar
variants in drosocin.
Acknowledgements We thank Department of Science and Technology
(project no.: SR/S1/OC-63/2012), India for financial support. We also
thank Ms. Shanta Sen for help with HRMS data.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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