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Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Coccinia indica agglutinin, a 17 kDa PP2 like phloem lectin: Affinity

purification, primary structure and formation of self-assembled
filaments
Kishore Babu Bobbili a , Gottfried Pohlentz b , Akkaladevi Narahari a,1 , Kaushal Sharma c ,
Avadhesha Surolia c , Michael Mormann b , Musti J. Swamy a,∗,2
a
School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
b
Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, 48149 Münster, Germany
c
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India

a r t i c l e i n f o a b s t r a c t

Article history: Phloem protein-2 (PP2) is an abundant soluble protein in the sieve elements in plants. Its lectin property
Received 28 September 2017 was reported in various species. The primary structure of a 17 kDa PP2 from Coccinia indica (Coccinia
Received in revised form 4 November 2017 indica agglutinin, CIA17), determined by mass spectrometry, shows extensive homology with PP2 super
Accepted 6 November 2017
family phloem lectins. Analysis of mass spectrometric data indicated the presence of 16 potential allelic
Available online xxx
variants of CIA17 with insignificant divergence in the primary structure. The primary structure contains an
intramolecular disulfide bridge between Cys-34 and Cys-51, which is conserved across various cucurbit
Keywords:
species and hence likely to be important for carbohydrate binding. CD spectroscopic studies revealed
Phloem protein 2
Mass spectrometry
that CIA17 is rich in antiparallel ␤-sheets, similar to PP2 proteins from Cucurbita maxima and Arabidopsis
Atomic force microscopy thaliana. CD spectra recorded at various temperatures showed very little change in the spectral intensity
and shape up to 90 ◦ C, suggesting that CIA17 is a highly thermostable protein. Atomic force microscopic
studies revealed that CIA17 forms filamentous structures at higher concentrations. In light of these results,
we propose that CIA17 and other PP2 proteins play a role in the plant defense against pathogens by directly
binding with the chitin cell wall, and also promote wound healing by forming self-assembled filaments.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction not known, their capacity to efficiently bind to exogenous carbo-

The phloem proteome is an integral component of the whole
plant communication system and also plays a crucial role in the
wound sealing and defense responses in the plants [1,2]. Chitin, a
polymer of ␤(1–4) linked N-acetylglucosamine, is a major compo-
nent of the fungal cell wall. Plants do not have chitin but possess
enzymes that can degrade it. The resulting fragments can be joined
together to form higher degree polymeric structures by chitinases
produced by pathogens, by a process called transglycosylation.
Also, non-catalytic chitin binding lectins present in pathogenic
fungi can protect chitin from degradation by plant chitinases [3].
Although the exact role of chitin binding lectins of plant origin is
∗ Corresponding author.
E-mail addresses: mjswamy1@gmail.com, mjswamy@uohyd.ac.in (M.J. Swamy).
1
Present address: Department of Biochemistry, University of Missouri, Columbia,
MO 65211, USA.
2
Website: http://chemistry.uohyd.ac.in/∼mjs/.
hydrate structures has led to speculations about their involvement
in defense against pathogens.
Phloem proteins (in short P-proteins) in the sieve tube were
identified as amorphous, crystalline, filamentous, tubular and fib-
rillar structures when observed by microscopy [4–6]. PP1 and PP2
are the most abundant phloem proteins in the sieve tube. While
PP1 family proteins with a Mr of ∼96 kDa, are exclusively found in
cucurbits [2,7,8], PP2 family proteins have been identified in many
angiosperms including celery and Arabidopsis and genome analysis
of Arabidopsis revealed that about 30 predicted genes code for pro-
teins that share homology with PP2 [9]. Multigene PP2 like phloem
lectins are common in many genera of plant kingdom [10]. In
Cucurbita maxima, PP2, a dimeric phloem protein with a molecular
weight of 48 kDa forms disulfide bonds with PP1 to form filamen-
tous structures [11]. Although lectin properties of some of these
proteins have been studied in detail in the past, the plausible roles
of these proteins and their natural ligands in the plant cells are not
known yet [9,11–17]. However, their binding specificity for chitin
and viroid RNA, expression in biotic and abiotic stress as well as

https://doi.org/10.1016/j.ijbiomac.2017.11.024
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their translocation and wound sealing properties suggest that they
probably play a role in defense against pathogens [11,18–20]. This
is further supported by their binding to exogenous carbohydrate
structures but not to endogenous ones [21–23].
The chitin binding phloem exudate lectins have been
reported to have more than one binding site for oligomers of
N-acetylglucosamine. They also bind to high-mannose type N-
glycans, most likely through the core GlcNAc␤14GlcNAc moiety
[14,15,24], and interact with viroid RNA in a nonspecific man-
ner in vivo, which has been revealed by intergeneric graft assays
[18,19]. Most phloem lectins which show homology with PP2 are
homodimers linked by intermolecular disulfide bonds. These pro-
teins show size polymorphism; mostly, an N-terminal peptide of
about 62 amino acids is absent in the 17 kDa lectins from cucum-
ber and melon as compared to the 24 kDa PP2 lectins present in the
other species such as pumpkin and ridge gourd. However, the rest
of the polypeptide containing the central and C-terminal segments
exhibit high homology. Preservation of lectin activity in all the pro-
teins clearly indicates that the N-terminal 62-amno acid sequence
is not essential for the carbohydrate binding activity [9].
Coccinia indica agglutinin is a homodimeric PP2 like lectin with
a subunit mass of 17 kDa isolated from the phloem exudate of
raw, unripe Coccinia indica fruits, commonly known as ivy gourds,
belonging to the Cucurbitaceae family [25]. In the present work,
besides the 17 kDa protein we identified the presence of two other
major chitin binding proteins with approximate molecular weights
of 24 and 10 kDa in the phloem exudate of Coccinia indica (ivy
gourd) fruits. Therefore, we refer to the 17 kDa chitin binding lectin
as CIA17. In the work reported here we determined the amino
acid sequence of CIA17 by mass spectrometry, investigated its
secondary structure and thermal stability using CD spectroscopy.
In addition, atomic force microscopic studies were employed to
demonstrate that CIA17 forms large filamentous aggregates at high
protein concentrations.
2. Materials and methods
2.1. Materials
Unripe ivy gourds (Coccinia indica fruits) were purchased
from local vegetable vendors. ␤-Chitin from squid pen (Sigma)
was a kind gift from Prof. A. R. Podile (School of Life Sciences,
University of Hyderabad). 2-Mercaptoethanol (␤ME), ␣-chitin, chi-
tooligosaccharides and thermolysin were purchased from Sigma
chemical Co. (St. Louis, MO, USA, and Taufkirchen, Germany,
respectively). Sodium chloride, disodium hydrogen phosphate and
sodium hydrogen phosphate were obtained from Merck India
(Mumbai). Micro Bio-Spin P6 columns were purchased from BIO-
RAD (Munich, Germany). Trypsin and chymotrypsin were obtained
from Roche Diagnostics GmbH (Mannheim, Germany). Methanol,
formic acid, acetic acid, dithiothreitol and iodoacetamide were
from Fluka (Buchs, Switzerland).
2.2. Purification of CIA17
CIA17 was purified by affinity chromatography on chitin. Ivy
gourds were washed thoroughly with distilled water, dried at room
temperature and bled by making long, 1–2 mm deep cuts on the
surface. The exudate that oozed from the cuts was collected imme-
diately into ice-cold PBS-␤ME (20 mM sodium phosphate buffer
containing 150 mM NaCl and 10 mM ␤ME, pH 7.2). The exudate
was centrifuged at 4 ◦ C for 20 min at 10,000 × g and the small pellet
discarded.
Affinity chromatography was performed on a column of ␣-chitin
(16 × 4 cm), prepared as described earlier [10,16]. In the first step,
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the phloem exudate collected in PBS-␤ME was directly loaded on
the affinity column and washed extensively with the same buffer.
Then the bound proteins were eluted with 0.1 M acetic acid. The
peak fractions were pooled and dialyzed against distilled water and
then loaded on a ␤-chitin column which was pre-equilibrated with
distilled water. After washing the column thoroughly with distilled
water until the absorbance at 280 nm of the washing reached 0.01
OD, the bound protein was eluted with 0.1 M acetic acid. SDS-PAGE
analysis revealed that only the 17 kDa protein is present in the elu-
ate. We refer to this protein as CIA17 (for Coccinia indica agglutinin
with a subunit mass of 17 kDa). Concentration of purified CIA17
was determined by the Bradford assay [26].
2.3. Mass spectrometry
2.3.1. Proteolytic digestion and acid hydrolysis
Prior to enzymatic cleavage 750 pmol of CIA17 was rebuffered
to 25 mM ammonium bicarbonate buffer by use of Biogel 6 spin
columns according to manufacturer’s instructions. Aliquots of
250 pmol each were reduced by 10 mM dithiothreitol (DTT) for 1 h
at 56 ◦ C. Subsequently, the reduced samples were alkylated under
exclusion of light in the presence of 55 mM iodoacetamide (IAA)
at room temperature. Samples were reconstituted again in 25 mM
ammonium bicarbonate buffer using Biogel 6 spin columns. Sub-
sequently, samples were thermally denatured at 95 ◦ C for 10 min.
In another set of experiments, equal amounts of CIA17 were sub-
mitted to proteolytic digestion without reduction and alkylation.
Reduced and native (unreduced) samples were incubated with
either trypsin or chymotrypsin overnight at 37 ◦ C with a substrate
to enzyme ratio of 25:1. A substrate to enzyme ratio of 12:1 was
employed when thermolysin was used as a protease and samples
were incubated overnight at 65 ◦ C. Then, the digests were dried,
redissolved twice in 150 ␮L of deionized water and dried in vacuo
and stored at −20 ◦ C. For chemical cleavage of CIA17, aliquots
(250 pmol) of either reduced and alkylated or untreated substrate
were reconstituted in deionized water as described above and dried
in vacuo. Then, samples were redissolved in 20 ␮L of deionized
water and 20 ␮L of 25% acetic acid and the mixture was incubated at
95 ◦ C for 2 h. Subsequently, samples were dried in vacuo and stored
at −20 ◦ C.
2.3.2. De novo sequencing
Nanoelectrospray mass spectrometry experiments were per-
formed using a quadrupole time-of-flight (Q-TOF) mass spectrom-
eter (Micromass, Manchester, UK) in the positive ion mode (ESI(+))
as described previously [27]. A Z-spray atmospheric pressure ion-
ization (API) source was used, with the source temperature set
to 80 ◦ C and a desolvation gas (N2 ) flow rate of 75 L/h. Home-
made nanospray capillaries were used, with the capillary tip set
to a potential of 1.1 kV and a cone voltage of 40 V. Gas-phase ions
were generated from solutions containing approx. 5 pmol/␮L ana-
lyte material in water/acetonitrile/formic acid (49/49/2, v/v/v). For
low-energy collision induced dissociation (CID) experiments, the
peptide precursor ions were selected in the first quadrupole ana-
lyzer and fragmented in the collision cell using a collision gas
(Ar) pressure of 3.0 × 10−5 mbar and collision energies of 20–40 eV
(Elab ). Peptide ions were fragmented by CID and their amino acid
sequences were deduced by evaluating the fragment ion series
(mainly b- and y-type ions) observed in the corresponding CID
spectra manually. Since the amino acids isoleucine (I) and leucine
(L) are isomeric they cannot be discriminated by means of low
energy CID experiments. Thus, they were – whenever possible –
assigned to I or L based on appropriate data base homology com-
parisons. Similarly, the masses of glutamine (Q) and lysine (K) differ
only by 0.036 Da and are not unambiguously distinguishable with
the Q-TOF instrument used. Thus, in general, amino acids exhibiting
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Fig. 1. A) SDS-PAGE of Coccinia indica phloem exudate proteins eluted from chitin affinity column. Lane 1) chitin binding proteins eluted from the ␤-chitin column. Three
bands corresponding to 24, 17 and 10 kDa are seen. Lane 2) molecular weight markers. The molecular weights in kDa are indicated against each band. B) SDS-PGE of purified
CIA17. Lane M) molecular weight markers. Lane CIA17) Coccinia indica agglutinin (CIA17). The standards used in both A and B are: bovine serum albumin (66 kDa), ovalbumin
(45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), ␣-lactalbumin (14.2 kDa),
aprotinin (6.5 kDa). For SDS-PAGE, the gels contained 5% acrylamide in the stacking and 12% acrylamide in the resolving gels. C) Non-denaturing PAGE. CIA17 yielded three
separated bands in different buffer conditions. Lane 1, in water; lane 2, in PBS; lane 3, in PBS-␤ME.

an increment mass of 128 Da are assigned to Q based on appropriate
data base homology comparisons, or to K unless proven by a tryptic
cleavage site. The protein sequence data reported in this paper will
appear in the UniProt Knowledgebase under the accession number
C0HK78.
2.4. Circular dichroism (CD) spectroscopy
CD spectral measurements were performed on a Jasco J-
810 spectropolarimeter. Wavelength scans were performed with
1.57 ␮M and 15.7 ␮M samples of CIA17 (monomer concentration)
for far and near UV regions, respectively. A rectangular quartz
cuvette of 0.2 cm pathlength was used for all measurements. Spec-
tra were recorded at a scan rate of 20 nm/min with a response time
of 4 s and a slit width of 2 nm. Measurements were made at differ-
ent temperatures between 30 and 95 ◦ C with the help of a Peltier
heating system connected to the instrument. Spectra at increasing
temperatures were recoded after incubating the sample at each
temperature for 5 min. A good signal-to-noise ratio was obtained
by averaging 5 consecutive scans collected from 190 to 250 nm
for far UV and 260–350 nm for near UV measurements at 1-nm
intervals. To investigate the effect of ligand on the conformation
of CIA17 the protein sample was incubated with 1 mM chitote-
traose overnight before recording the CD spectrum. Buffer scans
were recorded under identical conditions and were subtracted from
sample spectra before further analysis. In order to determine the
content of various types of secondary structures, the far UV CD spec-
trum of CIA17 was analyzed by three different methods, namely
CDSSTR, SELCON3 and CONTINLL [28], using the software routines
available at DICHROWEB (www.Cryst.bbk.ac.uk/cdweb/html) [29].
2.5. Atomic force microscopy
AFM images were obtained at room temperature using a SOLVER
PRO-M atomic force microscope from NT-MDT (Moscow, Russia)
as described earlier [30]. Aliquots (20 ␮L) from a freshly dialyzed
sample of CIA17 (1.5–57 in PBS-␤ME) were deposited on a freshly
cleaved mica surface (1 cm × 1 cm). The sample was air-dried on the
mica sheet for 10 min and gently washed twice with HPLC grade
water and again air-dried for 10 min. Then it was transferred to
the AFM stage, which was equipped with a 10 ␮m bottom scan-
ner. Imaging was performed in semi-contact mode. Images were
analyzed using NOVA software, supplied by NT-MDT.
2.6. Non-denaturing polyacrylamide gel electrophoresis
Ligand-induced oligomerization of CIA17 was investigated by
non-denaturing polyacrylamide gel electrophoresis (NDE). Exper-
iments were performed using continuous polyacrylamide gels
consisting of 10% (w/v) acrylamide in 25 mM Tris/250 mM glycine
buffer pH 8.3. Samples were prepared by mixing 10 ␮L of CIA17
(34.6 ␮M) and a 10 ␮L aliquot of 2× sample buffer containing bro-
mophenol blue. The samples were then loaded on the gel and
subjected to electrophoresis. Proteins were visualized by staining
the gels with Coomassie Brilliant Blue R-250.
2.7. Cysteine modification
The free cysteine residues in CIA17 were alkylated by chemical
modification as reported earlier [11]. CIA17 was dialyzed against
Tris-HCl buffer (pH 8.4) containing 25 mM DTT to reduce the disul-
fide bonds and the resulting free thiol groups were modified by
adding 60 mM iodoacetic acid followed by incubating the mix-
ture under exclusion of light for 2 h at 4 ◦ C. The cysteine modified
CIA17 (CMCIA17) was dialyzed thoroughly against distilled water
to remove excess reagent. Concentration of the protein was esti-
mated by the Bradford assay [26].
3. Results
3.1. Affinity chromatographic purification of CIA17 and
macromolecular characterization
When the phloem exudate of ivy gourds was subjected to affin-
ity chromatography on ␣-chitin in PBS-␤ME, three proteins with
approximate subunit masses of 24 kDa, 17 kDa and 10 kDa were
bound to the affinity matrix (Fig. 1A). When these proteins were
eluted and their mixture was subjected to a second affinity chro-
matographic step using ␤-chitin as stationary phase, only the
17 kDa protein (CIA17) was retained on the column, and the 10 and
24 kDa proteins did not bind to the matrix (Fig. 1B). Interestingly,
in native PAGE (Fig. 1C), CIA17 showed multiple bands indicating
the presence of oligomeric forms (or soluble aggregates) that differ
in size. This was further investigated by atomic force microscopy
(see below). Activity of the 17 kDa protein (CIA17) was assessed
by the hemagglutination assay and its concentration was deter-
mined spectrophotometrically using an A280 nm value of 3.08 for a

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Fig. 2. Amino acid sequence of CIA17 derived from mass spectrometric experiments and its alignment with the primary structures of other 17 kDa PP2 proteins from
Cucurbitaceae. The proteins are, from top to bottom: CIA17, Cucu17 (Q8LK70 17 kDa phloem lectin [Cucumis sativus]) and Cmm17 (AF517156 1 17 kDa phloem lectin Lec17-1
[Cucumis melo]). Multiple alignments were established with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The predicted carbohydrate binding regions in the
sequences are shown in red boxes (residues shown in blue). The cysteine residues (C34 and C51 in CIA17, shown in green) involved in the intramolecular disulfide linkage
are indicated by green arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

1.0 mg/mL sample for 1-cm path length, calculated from its primary
structure (see below) as described previously [31], and expressed
either in mg/mL or in terms of monomer (Mr = 17,495 Da, estimated
from its primary structure, determined by mass spectrometry, as
discussed below).
3.2. Primary structure of CIA17 – de novo sequencing
The amino acid sequence of CIA17 was derived by de novo
sequencing of peptide precursor ions obtained from in-solution
proteolysis using trypsin, chymotrypsin and thermolysin as pro-
teases and by use of chemical proteolysis employing acetic acid.
Sequences were deduced from fragment ion spectra and the respec-
tive sequence stretches were aligned to the sequence reported for
a 17 kDa phloem lectin obtained from Cucumis sativus (cucum-
ber) (sequence entry: Q8LK70 CUCSA) giving rise to a coverage of
almost 100% of the reported 154 amino acids (Fig. 2). A synopsis of
the respective sequenced peptide ions matching variants of CIA17
along with their experimental and calculated m/z values is given in
Supplementary Table S1. A representative example of a low energy
CID mass spectrum obtained by activation of doubly charged tryp-
tic peptide ions with m/z 1324.65 is shown in Fig. 3. A complete set
of y-type ions comprising the C-terminus of the peptide precursor
accompanied by some complementary b-type fragment ions allows
for assignment of the amino acid sequence. Evaluation of sequence
data indicated the presence of microheterogeneity within CIA17 to
some extent viz. combination of the different peptide isoforms leads
to 16 possible distinct species (Fig. 4). An intramolecular disulfide
bridge was also determined by the use of low energy collision-
induced dissociation (CID) experiments. Fragmentation of doubly
charged disulfide bond-containing peptide ions with m/z 955.92
gave rise to proton-induced asymmetric cleavages of the disulfide
bridge preceding rupture of the peptide backbone (Fig. 5), which
enabled the determination of both amino acid sequence and the
position of the disulfide bond as described previously [32]. The
complete sequence and its alignment with that of other known
17 kDa PP2 proteins are shown in Fig. 2.
3.3. CD spectroscopic studies: secondary structure and thermal
stability of CIA17
The CD spectrum of CIA17 in the far UV region shows a nega-
tive band centered at 217 nm and two positive bands at 230 and
200 nm (Fig. 6A, solid line). The strong negative band between 210
and 220 nm and the positive band near 200 nm are characteristic
for the presence of ␤-sheets as the predominant conformation in
the protein [33]. Analysis of the CD spectra by 3 different meth-
ods yielded the content of various types of secondary structures as:
42.2% ␤-sheets, 30% ␤-turns, 24.6% unordered structure and 2.6% ␣-
helix. Only minor changes were observed in the presence of 1 mM
chitotetraose (Fig. 6A, dashed line), indicating that carbohydrate
binding does not significantly alter the secondary structure of the
protein. The near UV CD spectrum of CIA17 (Fig. 6B) is characterized
by a broad intense peak at 280 nm and a peak of slightly lower inten-
sity at ∼288 nm, which could be attributed to the absorption of the
side chains of tyrosine and tryptophan residues, respectively [34].
This is consistent with the presence of 4 tyrosine and 9 tryptophan
residues in the primary structure of CIA17 (Fig. 1).
The near UV CD spectra of CIA17 alone and in presence of 1 mM
chitotetraose at 25 ◦ C are shown in Fig. 6B. Moderate changes are
observed in the spectral intensity when the spectrum was recorded
in the presence of 1 mM chitotetraose; these changes are con-
sistent with a slight compaction of the overall protein structure
due to ligand binding. Since the association constant for the inter-
action of chitotetraose with CIA17 was previously estimated as
6.5 × 105 M−1 , at this ligand concentration the protein is expected
to exist essentially in the ligand bound state [35].
Far and near UV CD spectra of CIA17 recorded at various tem-
peratures between 30 and 95 ◦ C are shown in Fig. 6C and D. Very
minor changes are seen in the spectral pattern in both these regions
up to 80 ◦ C, whereas moderate decrease in the spectral intensity is
observed at 90 ◦ C, which decreases further at 95 ◦ C. However, con-
siderable spectral intensity was retained even at 95 ◦ C, indicating
that the protein is only partially unfolded. In addition, the spectral
band shape was not significantly affected even at 95 ◦ C, indicating
that the antiparallel ␤-sheet structure is retained at all tempera-
tures, although its relative proportion decreases as the temperature
is increased. These observations suggest that CIA17 is a highly ther-
mostable protein.
3.4. Direct imaging of CIA17-chitooligosaccharide interaction by
AFM
To investigate if CIA17 forms aggregated structures at high con-
centrations, we performed AFM studies. AFM images of native
CIA17 obtained at low sample concentration (1.57 ␮M) show glob-
ular particles distributed uniformly (Fig. 7A) with a height per
particle of ∼15 nm. Interestingly, when the CIA17 concentration

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Fig. 3. A) NanoESI Q-Tof fragment ion spectrum obtained from a low-energy CID experiments on the doubly charged precursor peptide ions with m/z 1324.65 derived from
an in-solution proteolytic digestion of reduced and alkylated CIA17 (aa: 25–45) using trypsin as protease. B) Corresponding fragmentation scheme (CCAM : carboxymethylated
cysteine).

was varied between 5.7 and 57 ␮M, filamentous structures of more
than 150 nm length were observed (Fig. 7, panels B–D). Impor-
tantly, density of the filamentous aggregates is seen to increase
with increasing protein concentration.
4. Discussion
In response to stress by phloem feeding insects, plants defend
themselves by using the phloem based defense mechanism which
can also be activated by wounding and oxidative conditions besides
insect attack [36,37]. Analysis of the recently released cucum-
ber genome has facilitated the unraveling of many interesting
aspects of phloem anatomy and its functions in plants [38]. For
a long time, Cucurbitaceae plants (cucurbits) have been investi-
gated extensively as they were considered to be very good models
to study phloem physiology in plants; recent work has clearly
shown that there are two independent transport systems in the
phloem, namely fascicular phloem (FP) transport system and extra
fascicular phloem (EFP) transport system. The well-studied phloem
proteins PP1 and PP2 are usually absent or present in very low
concentrations in the FP, but are abundant in EFP [39]. The wide
distribution of PP2 like genes in plant kingdom indicates that in
addition to Cucurbitaceae species they are present in a variety of
other plants [9]. PP2 proteins are being investigated in detail in
view of their carbohydrate and RNA binding properties as well as
the translocation of the ribonucleoprotein complexes formed by
them [15,18,19,40]. The ability of PP2 proteins to transport a range
of macromolecules throughout the plant led to speculations regard-
ing their role in interacting with other proteins [2]. Understanding
structure-function relationships of proteinaceous filaments in sieve
elements has long been a subject of inquiry in phloem biology
[5,6,11]. The highly abundant P-proteins PP1 and PP2 are thought
to be involved in the formation of filaments; PP1 proteins can
form filaments as well as gel at higher concentrations by forming
reversible oxidative disulfide cross links with PP2 [5]. Therefore PP2
proteins also appear to be essential components in the formation
of filaments/soluble aggregates besides their carbohydrate binding
property.
The presence of a homodimeric phloem lectin with a
subunit mass of 16 kDa in Coccinia indica (Coccinia indica agglu-

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Fig. 4. Amino acid sequences of the 16 allelic variants of CIA17 whose primary structures could be assigned with confidence are shown with sequence alignment.
tinin, CIA) was reported previously by Surolia’s group [25].
Its chitooligosaccharide-binding characteristics and tryptophan
involvement at the binding site were investigated by fluores-
cence spectroscopy [25,41]. We found the molecular weight of
this protein to be ∼17.5 kDa by mass spectrometry-based de novo
sequencing experiments; hence we refer to it as CIA17. Bands cor-
responding to 24 kDa and 10 kDa proteins were also identified in
the SDS-PAGE analysis of fractions obtained from chitin affinity
chromatography (Fig. 1A), similar to the observations made with
the chitin-binding proteins in the phloem exudate of Cucumis melo
[19]. The 24 kDa band is most likely a PP2 like phloem lectin and
the 10 kDa band probably corresponds to an uncharacterized chitin
binding protein. Further work to characterize these proteins is cur-
rently underway in our laboratory.
4.1. Amino acid sequence and homology with other PP2 proteins
The amino acid sequence of CIA17, derived from mass-
spectrometric studies, shows high homology with well-known
17 kDa PP2-like phloem lectins from Cucumis sativus (cucumber)
and Cucumis melo (melon) (Fig. 2), suggesting that it is also a PP2
like phloem lectin. The presence of 16 possible allelic variants with
insignificant divergence in the primary sequence of CIA17, similar
to the 17 kDa PP2 protein from C. sativus suggests that multigene
PP2 families are common in angiosperms [9]. But the functional
divergence of PP2 proteins of allelic variants in the same species
is not known, though their lectin activity remains the same. PP2-
like phloem lectins can also interact with nucleic acids [18,19,40].
In this respect PP2 proteins are similar to histone proteins, another
group of multigene proteins which interact with DNA to form stable
Please cite this article in press as: K.B. Bobbili, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.11.024
nucleosomes [42,43]. Another similarity of PP2 proteins with his-
tone proteins is in having a unique N-terminal length and sequence
along with a conserved C-terminal segment [9]. The primary struc-
ture of CIA17 contains 3 cysteine residues; while C34 and C51 form
an intramolecular disulfide bond, cysteine C125 which is near the C-
terminal region, is probably exposed on the surface and takes part
in the formation of an intermolecular disulfide linkage. Employ-
ing a mutational approach, we have recently identified the amino
acid residues involved in carbohydrate binding by pumpkin phloem
lectin (PPL), which are highly conserved in PP2 proteins from Cucur-
bitaceae [44]. Interestingly these residues are also conserved in
CIA17 (Fig. 2). Additionally, these residues are positioned in a short
peptide segment whose conformation is locked by an intramolec-
ular disulfide linkage between Cys-34 and Cys-51, suggesting that
this disulfide bond is crucial for carbohydrate recognition by PP2.
4.2. CD spectroscopic studies on the secondary structure and
thermal stability of CIA17
The intense positive band around 195–200 nm in the far UV CD
spectrum of CIA17 indicates the presence of antiparallel ␤-sheet
as the predominant secondary structure (Fig. 6A). This has also
been shown in the model structures of other PP2 like proteins
from Arabidopsis and pumpkin [15,24]. Similarities in the amino
acid sequence and structural features of PP2 proteins with carbo-
hydrate binding modules (CBM) indicate that all of them contain
␤-sheets as the major secondary structure [15,24,45].
Binding of chitooligosaccharides does not bring about any major
changes in the far UV CD spectrum of CIA17, clearly indicating that
protein structure is retained even after binding to the ligands with
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Fig. 5. A) NanoESI Q-Tof fragment ion spectrum obtained from a low-energy CID experiments on the doubly charged precursor peptide ions with m/z 955.92 derived from
an in-solution proteolytic digestion of CIA17 (aa: 27–36 and 49–53 joined by a disulfide bridge) using chymotrypsin as protease. B) Corresponding fragmentation scheme.

high affinity (Fig. 6A). It has been shown previously that CIA17 has
extended binding sites which can accommodate up to a tetrasac-
charide. Besides this, presence of the fourth sugar residue proximal
to the highly fluorescent tryptophan at the binding site leads to
a blue shift in the fluorescence emission maximum [25]. Similar
chitooligosaccharide-induced blue shift in the fluorescence emis-
sion maximum of the protein was also reported for Luffa acutangula
agglutinin, another chitooligosaccharide-specific lectin [14]. These
results imply that tryptophan residues are important for the car-
bohydrate binding by these lectins.
Results of temperature dependent CD spectroscopic studies on
CIA17 indicate that the protein retains most of the secondary and
tertiary structures up to 90 ◦ C, suggesting that the protein is highly
thermostable. Interestingly, even the spectrum recorded at 95 ◦ C
showed considerable intensity in the bands at ∼200 and 215 nm,
clearly indicating the presence of considerable secondary structure.
These results show that CIA17 is more thermostable than two other

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Fig. 6. A) Far- and B) near-UV CD spectra of CIA17 in the absence (——) and in the presence (----) of 1 mM chitotetraose. Thermal stability of CIA17 is investigated by recording
the CD spectra at various temperature in the C) far-UV and D) near-UV CD regions. Protein concentration for measurements in the far and near UV regions was 1.48 ␮M and
14.8 ␮M, respectively. Buffer: 20 mM PBS-␤ME (pH 7.2). Spectra were recorded at: 30, 40, 50, 60, 70, 80, 90 and 95 ◦ C. Cell path length was 2 mm.

Fig. 7. AFM images of CIA17 at different sample concentrations. A) 1.57 ␮M, B) 5.7 ␮M, C) 20 ␮M, D) 57 ␮M. Size of each image is 5 × 5 ␮m.

phloem lectins from Cucurbitaceae, namely SGPL and PPL, which
were found to unfold around 73 ◦ C and 82 ◦ C, respectively [16,46].
4.3. Filament formation by CIA17 – role in wound sealing
Previously PP2 proteins have been proposed to play a signif-
icant role in closing wounded parts of the plant resulting from
predator incision [11,47]. The presence of multiple bands in native
PAGE (Fig. 1C) suggested that CIA17 forms oligomeric structures
in the native state. To investigate the nature of such aggregates
we carried out AFM studies. It may be noted that P-protein orga-
nization and filament formation was observed in situ by TEM in
Arabidopsis [48]. Our results from AFM studies indicate that while
CIA17 probably forms small globular soluble aggregates at low con-
centration (Fig. 7A), the globular aggregates appear to associate
further to yield filamentous structures at intermediate concen-
trations (Fig. 7B, C). whereas at high concentration the filaments
aggregate together yielding large bundles (Fig. 7D). To the best of
our knowledge, this is the first report of direct imaging of such fil-
amentous aggregates formed by the PP2 proteins from the phloem
exudate of Cucurbitaceae species. Cross-linking of such filamen-
tous structures/bundles made up of PP2 proteins with PP1 proteins
are likely to be involved in the wound sealing in the Cucurbitaceae
fruits, as suggested by Read and Northcote [13].
In summary, this study reports the primary structure of CIA17,
a chitooligosaccharide-specific phloem exudate lectin that belongs
to the PP2 family of proteins. An intramolecular disulfide linkage
between Cys-34 and Cys-51, which is conserved across other PP2
proteins was found to lock the conformation of residues involved
in carbohydrate binding. The predominant secondary structure of
CIA17 is antiparallel ␤-sheets, which is similar to that found in other
PP2 proteins, such as those from Cucurbita maxima and Arabidopsis
thaliana. CD spectra recorded at various temperatures suggest that
CIA17 is a highly thermostable protein with an unfolding tempera-
ture above 90 ◦ C. AFM and non-denaturing electrophoresis studies
revealed that CIA17 forms filamentous structures at high concen-
trations, which could be important for wound healing in the plant.
Author contributions
KBB, AN and GP did all the experiments with help from KS. MJS,
MM, and AS conceived the ideas, planned the experiments and
supervised the research. KBB, MJS and MM wrote the manuscript.
All authors read and corrected 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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Acknowledgments
This work was supported by a research project from the Depart-
ment of Biotechnology (India) to MJS. We thank the University
Grants Commission (India) for its support through the UPE-II grant
to the University of Hyderabad and the CAS program to the School
of Chemistry. Support from the Department of Science and Tech-
nology under the FIST program is gratefully acknowledged. KBB
and AN were supported by Senior Research Fellowships from the
Council of Scientific and Industrial Research (CSIR), India. Financial
support by the Deutsche Forschungsgemeinschaft (DFG) for IRTG-
MCGS [Grant no. GRK 1549] is gratefully acknowledged. AS is a
Bhatnagar Fellow of the CSIR (India).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.ijbiomac.2017.11.
024.
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