A sunflower lectin with antipathogenic properties and putative pharmacological
applications
Mariana Regente1, Mariângela Diz2, Marcela Pinedo1, Mercedes Elizalde1, Valdirene Gomes2,
Laura de la Canal1.
1
Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata - CONICET, Funes 3250,
7600 Mar del Plata, Argentina, E-mail: ldelacan@mdp.edu.ar
2
Universidade Estadual do Norte Fluminense, Laboratório de Fisiologia e Bioquímica de
Microrganismos, Campos dos Goytacazes, 28013-602, RJ, Brazil, E-mail: valmg@uenf.br
ABSTRACT
● Lectins are carbohydrate-binding proteins with high specificity for a variety of sugar motives in
glycoconjugates. This characteristic supports the cell agglutination, antitumoral, immunomodulation,
antiviral, antibacterial, antifungal and insecticidal activities associated to certain lectins. Among them, the
jacalin-related lectins (JRLs) are considered to be a small sub-family composed of galactose and
mannose-specific members. Using a proteomic approach we have detected in sunflower seedlings a 16
kDa protein (Helja) which was further purified by mannose-agarose affinity chromatography.
● The aim of this work was to characterize the biological activity of Helja and to explore its potential
applications.
● To assess agglutinantion properties, yeast (Saccharomyces cerevisiae) cells were incubated with
growing concentrations of the purified protein. Helja clearly agglutinated these cells at a concentration of
120 μg/ml. Its carbohydrate-specificity was determined on the basis of the ability of different sugars to
inhibit cell agglutination. Among the monosaccharides tested, D-mannose showed the greatest inhibitory
effect, being 1.5 mM the minimal concentration required to avoid agglutination. These results confirm
that Helja is a mannose specific lectin. The antifungal activity of this JRL was evaluated using human
pathogenic fungi belonging to Candida sp. We show that 100 μg/ml of Helja partially inhibited the
growth of Candida albicans and induced morphological changes on Candida tropicalis through
pseudohyphae formation. On the other hand, the potential insecticidal effect of Helja against the bruchid
Callosobruchus maculates was explored. To that aim Helja was coupled to fluorescein isothiocyanate
(FITC) and its binding to larvae digestive tract was determined by fluorescent microscopy. The visualized
Helja-FITC signals revealed the interaction of the jacalin with carbohydrate moieties on the tissue surface.
● We concluded that Helja is a new member of the mannose-binding JRLs with cell agglutination
capability, antifungal activity against human pathogenic fungi and potential insecticidal properties. Novel
activities of Helja continue to be investigated.
● A novel sunflower lectin is described, whose biological properties may find practical applications to
control of pathogens.
Key words: Antifungal, insecticidal, jacalin, seed, sunflower.
INTRODUCTION
Plant derived medicines are widely spread and increasing in both traditional and modern pharmacology
(Canter et al, 2005). Lectins have been documented as one of the plant bioactive compounds against
pathogens of diverse origins. They are carbohydrate-binding proteins that recognize specific sugar
structures of glycoconjugates occurring in cell surface or in solution. This characteristic supports the cell
agglutination, antitumoral, immunomodulation, antiviral, antibacterial, antifungal and insecticidal
activities associated to this family of proteins which could find important practical applications (Lam and
Ng, 2011). Among them, the jacalin-related lectins (JRLs) are a sub-group composed of galactose and
mannose-specific members (Van Damme et al, 1998), which display relevant antipathogenic activities.
Precisely, this family comprises all the lectins that share sequence similarity with the agglutinin from jack
fruit (Artocarpus integrifolia), characterizated by its high specificity by the T-antigen (Sastry et al, 1986).
Using a proteomic approach we have detected in the extracellular fluids of sunflower seedlings, a 16 kDa
protein (Helja) predicted to be a mannose specific JRL through bioinformatic tools. Thus, mannoseagarose affinity chromatography allowed to obtain a purified fraction of Helja, whose identity was
confirmed by MALDI-TOF spectrometric assays (Pinedo et al, 2011).
In this work, we have assessed the potential applications of the sunflower jacalin-related lectin Helja in
the control of pathogens through the characterization of its biological activity against a group of selected
organisms.
MATERIALS AND METHODS
Plant material and collection of extracellular fluid
Helianthus annuus L seeds (line 10347 Advanta Semillas) were imbibed for different times or grown
under conditions previously described (Pinedo et al, 2011) and subjected to the extraction of the
extracellular fluids (EF) by a standard infiltration-centrifugation procedure (Regente et al, 2009). Briefly,
seeds or seedlings were immersed in 50 mM Tris-HCl pH 7.5, 0.1 % NaCl, 0.1% 2-mercaptoethanol and
subjected to three vacuum pulses of 10 seconds, separated by 30 seconds intervals. The infiltrated
materials were recovered, dried on filter paper, placed in fritted glass filters and centrifuged for 20 min at
400g at 4ºC. The EF was recovered in the filtrate and evaluated for the absence of intracellular
contamination as previously described (Regente et al, 2009).
Helja purification and protein analysis
The purification of Helja was performed according to Pinedo et al. (Pinedo et al, 2011), with some
modifications. Ten ml of EF obtained from seeds were loaded on a 1 ml D-mannose-agarose resin (Sigma
M6400) equilibrated with buffer A (50 mM HCl-Tris pH 7.5, 100 mM NaCl). Non-bounded proteins were
washed exhaustively before the elution of retained proteins with 4 ml of 0.2 M mannose in buffer A. The
eluted fraction was loaded on a centrifugal filter device centricon YM 3 to allow the separation of the
protein fraction from mannose.
Electrophoretic separation was performed in 12% SDS-PAGE gels by standard methods. When indicated,
this procedure was followed by the transfer the proteins to 0.45 µm nitrocellulose membranes. For
Western blot analysis the membranes were incubated in blocking buffer followed by the anti-Helja
antiserum diluted 1/1.000.
Agglutination assay
The agglutination assay was performed using yeast (Saccharomyces cerevisiae) cells due to the easy of
growth and cultivation. The assay was carried out on micro slides containing increasing concentrations of
the lectin and 5 µl of yeast cells diluted 1/100 (Undiluted cell cultures displayed OD=0.25). After 15 min
of incubation samples were microscopically evaluated. Controls were performed replacing the lectin
sample with the same volume of water. The inhibition of cell agglutination was evaluated by the addition
of different concentrations of mannose, galactose, glucose or fructose to the incubation mix.
Effect of Helja on yeast growth
For the preparation of Candida albicans and Candida tropicalis cell cultures, an inoculum of each yeast
was transferred to Petri dishes containing agar Sabouraud and allowed to grow at 28 ºC for 3 days. After
this period, cells were transferred to sterile Sabouraud broth (1 mL). Yeast cells were quantified in a
Neubauer chamber for further calculation of appropriate dilutions. To monitor the effect of Helja on the
growth of yeasts, cells (104 in 1 ml Sabouraud broth) were incubated in the presence of the 100 μg/ml at
28 ºC in 200 μl microplates. The spectrophotometer was blanked with culture medium alone then optical
readings at 670 nm were taken at the zero time point and then every 6 h for the following 36 h (Broekaert
et al, 1990). After 30 h of the yeast growth inhibition assay, yeast cells were separated from the growth
medium using centrifugation, washed in Sabouraud broth and plated for observation in an optical
microscope at 400 X magnification (Axiovert 135).
Covalent conjugation of FITC to Helja and binding to insect digestive tract
FITC (Fluorescein Isothiocyanate) was covalently coupled to Helja and, additionally, to egg albumin as a
control. FITC (50 mg in 1 mL of anhydrous DMSO) was immediately diluted in 0.75 M bicarbonate
buffer (pH 9.5) before use. Following the addition of FITC to give a ratio of 1 mg FITC per mg of lectin
and egg albumin, the tube was wrapped in foil and incubated in an orbital shaker at room temperature for
1 h. Unreacted FITC was removed by dialysis against distilled water, and the resulting solution was
freeze-dried. After 20 days in the artificial seed system, larvae were removed, and their intestine,
malpighian tube and trachea were dissected and collected together. These materials were treated with 10
µg of the Helja-FITC and albumin-FITC complex. After the treatments, the materials were washed with
PBS, mounted on glass slides and visualized using a DIC microscope (Axiophoto Zeiss) equipped with a
fluorescence filter set for fluorescein detection (excitation wavelengths, 450 to 490 nm; emission
wavelength, 500 nm) for detection of labeled lectin.
RESULTS AND DISCUSSION
The apoplastic fraction of germinating seeds and seedlings were isolated and controlled for the absence of
intracellular contaminants (Regente et al, 2009). Subsequently they were analyzed for the relative
abundance of Helja along different stages of development using a Western blot approach. Figure 1 shows
that Helja content decreased through the germination and seedling growth being almost undetectable in 6
days-old plants. Since 2 h imbibed seeds yielded the highest amounts of the lectin their extracellular
fluids were subjected to D-mannose affinity chromatography to purify this jacalin. In agreement with in
silico assigned binding properties and the previous results from sunflower seedlings (Pinedo et al, 2011),
a protein of 16 kDa, the molecular mass expected for Helja, was retained by the mannose matrix (Figure
2). In order to confirm its identity, it was automatically recovered from the gel, digested with trypsin and
submitted to a MALDI-TOF spectrometric analysis. The obtained peptide mass fingerprint matched to the
Helianthus tuberosus JRL Q9FS32 (Uniprot accesion) sequence and confirmed the identification (not
shown).
Figure 1
Helja levels in apoplastic fluids of sunflower germinating seeds. Extracellular proteins from 2 hours (2h),
1 (1d), 2 (2d), 4 (4d), 5 (5d) or 6 days (6d) germinating seeds were loaded on a 12% SDS-PAGE and
subjected to Western blot analysis using 1:1000 anti-Helja serum. Molecular weight markers are indicated
on the left.
kDa
37
25
20
15
2h
1d
2d
4d
5d
6d
Figure 2
Gel electrophoretic analysis of the purified Helja. Apoplastic fluid from sunflower seeds was fractionated
by mannose affinity chromatography. 1: Non-retained fraction. 2: Mannose-eluted fraction. Molecular
mass markers are indicated on the left.
kDa
1
2
250
148
98
64
50
36
22
HelJa HelJa
16
6
4
To characterize the biological activity of Helja we explored its ability to agglutinate Saccharomyces
cerevisiae cells, known to be enriched in a mannan oligosacharide in their cell wall. Helja produced a
clear agglutinating effect at 120 μg/ml while 1.5 mM D-mannose was found to inhibit this action (Figure
3). The evaluation of the specificity and affinity for carbohydrate binding was performed by adding
different concentrations of particular sugars to the agglutination mix and the minimum inhibitory
concentration (MIC) was estimated. Since mannose showed the most potent inhibitory capacity (MIC 1.5
mM) the preference of the sunflower jacalin for this carbohydrate was concluded. Other sugars as
galactose, glucose and fructose were 2-10 times less inhibitory than mannose (Table 1).
Figure 3
Effect agglutinant of Helja on Saccharomyces cerevisiae cells under microscopic observation. (A), in the
absence of Helja; (B), in the presence of 120 μg/ml of Helja; (C), in the presence of 120 μg/ml of Helja
and 1.5 mM of mannose.
A
B
C
Table 1
Sugar specificity of Helja. Inhibition of Saccharomyces cerevisiae agglutination was determined by
adding growing concentrations of the indicated sugars at a Helja concentration of 120 μg/ml. The lowest
concentration of sugars that inhibit agglutination was defined as MIC (minimum inhibitory concentration).
Sugar
Mannose
Galactose
Glucose
Fructose
MIC (mM)
1.5
5.0
10
10
To further characterize potential applications of Helja its effect on the human pathogens Candida
albicans and Candida tropicalis was evaluated. C. albicans growth was only partially inhibited by the
protein and nearly unaffected growth values were observed in C. tropicalis cells employing
concentrations up to 100 μg/ml of jacalin (not shown). However, photomicrographs of C. tropicalis
showed morphological alterations, with the apparent formation of pseudohyphae, which were absent in
the control tests (Figure 4). This effect is similar to that reported for certain antimicrobial peptides (Stotz
et al, 2009). A cellular agglutination was also observed for Candida albicans yeasts, while normal growth
and development were observed for all control cells.
The potential insecticidal effect of Helja against the bruchid Callosobruchus maculatus was also explored.
To that aim the sunflower lectin was coupled to fluorescein isothiocyanate (FITC) and its interaction with
larvae digestive tract was evaluated. Fluorescent microscopy allowed the visualization of the Helja-FITC
signals on the trachea surface, indicating that the protein is able to bind carbohydrate moieties present in
this tissue (not shown for briefness). Although the precise mode of insecticidal action of plant lectins is
not fully understood it appears that resistance to proteolytic degradation by the insect digestive enzymes
and binding to insect gut structures are two basic prerequisites for lectins to exert their deleterious action
on insects. This effect has already been reported for other purified lectins (Coelho et al, 2007; Macedo et
al, 2007).
Figure 4
Antifungal effect of Helja on Candida albicans and Candida tropicalis cells. Photomicrographs of C.
albicans (A,B) and C. tropicalis (C,D) were taken after 30 h of yeast growth. (A,C) control (absence of
Helja); (B,D) presence of Helja (100 μg/ml). The arrow indicates a pseudohyphae.
A
B
C
D
In the present work evidences of cell agglutination ability, antifungal activity against human pathogenic
fungi as well as potential insecticidal effect of the sunflower protein Helja were presented. These
properties point out Helja as a good, naturally occurring, product which may be usefull for the biocontrol
of pathogens, a current area of intense research. Meanwhile, novel biological activities of this lectin
continue to be investigated.
ACKNOWLEDGMENTS
This work was supported by grants from the ANPCYT, CONICET and the University of Mar del Plata,
Argentina. We also acknowledge the financial support of the Brazilian agencies CNPq, CAPES and
FAPERJ.
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