SYNOPSIS

Name: Ms. Summra Ahmed

Father's Name: Mr. Nisar Ahmed Awan

Registration for: M. Phil.

Supervisor Name: Dr. Saima Rasheed

Assistant Professor
Dr. Panjwani Center for Molecular Medicine and Drug Research
International Center for Chemical and Biological Sciences (ICCBS)
University of Karachi, Karachi-75270, Pakistan.

Co-supervisors Name: a) Prof. Dr. M. Iqbal Choudhary, H.I., S.I., T.I.

Distinguished National Professor / Director
H. E. J. Research Institute of Chemistry
Dr. Panjwani Center for Molecular Medicine and Drug Research.
International Center for Chemical and Biological Sciences (ICCBS),
University of Karachi, Karachi-75270, Pakistan.

b) Prof. Dr. rer. nat. Christian Betzel

Head of the group
Institute of Biochemistry and Molecular Biology
Martin-Luther-King-Platz 6
20146 Hamburg, Germany

Topic of Research: Purification, Crystallization and X-Ray Structure Analysis of Plant

Lectins In-complex with Different Ligands.

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 Content

S. No. Topic Page No.

1. Abstract 3

2. Introduction 4-5

3. Hypothesis 5

4. Expected outcomes 6

5. Limitations 6

6. Literature survey 7-9

7. List of proposed drugs to be evaluate during study 9

8. Proposed work plan 10

References
11-16
Annexure-І

ABSTRACT
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Since last five decades, medicinal plants have attracted huge attention mainly due to their diverse
biological and therapeutic properties. Medicinal herbs and their constituents are frequently used
in various traditional, complementary, and alternative systems against different diseases. Plants
are rich source of phytochemicals and proteins, but among them proteins are now focussed for
medicinal applications, because of their bio- and cyto-compatiblity. Challenges of human health
care and sustainable development demands to utilize plant proteins for the treatment of different
ailments. For example, extracts of lectin- rich mistletoe (Viscum album) has been used for
treating cancer since 1920s. Nowadays, due to resistance of micro-organisms against
antimicrobial agents, it is a challenge to treat infections. Synergistic effect of two compounds
could be a solution to such resistant microorganisms. Hence, there is an increased interest in
antibiotic adjuvant therapy. Some commonly used combinations are Augmentin (Amoxicillin-
clavulanic acid), Timentin (ticarcillin-clavulanate), Septra (sulfamethoxazole- trimethoprim.),
and Bactrim (Trimethoprim-sulfamethoxazole). In this domain bioactive proteins, such as
lectins, may play an important role (as being natural antimicrobial agents) to enhance the
antimicrobial activity of the drugs/leads.
Lectins, a well-known class of carbohydrate binding proteins, are important for a variety of
biological processes through their ability to specifically bind with different sugars. Lectins
possess different biological activities such as anti-tumor, immunomodulatory, insecticidal, and
most importantly antimicrobial activities. Lectins mediate the identification of micro-organisms
through interaction with the carbohydrates present on microbial cell surfaces. Resistance, a major
challenge and global health care crisis in the 21st century, has emerged to all clinically used
antibiotics. Unfortunately there is a growing gap between need for new antibiotics and drug
discovery and development. Therefore, interest has been developed to investigate the role of
lectins as antimicrobial agents or to use it as adjuvant. We hypothesize that one of the best
choices for the antimicrobial activity is to evaluate lectin as adjuvant in combination with
antimicrobial drugs or leads.
Considering biological importance of lectins, we aim to isolate lectins from banana fruit (Musa
acuminata) and jack bean seeds (Canavalia ensiformis) by using affinity chromatography.
Additionally, proposed lectins will also be crystallized in apo as well as in-complex with
different antimicrobial drugs/compounds and sugars. In the last stage, structure analysis will be
carried out though state of-the-art X-ray crystallography. We hope that the outcome of these
studies will result in the discovery of safe and effective adjuvant anti-microbial therapy that will
serve as leads to improve the quality of human life.

INTRODUCTION

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Discovery of antibiotics has not only revolutionized the drug discovery process but also resulted
in a dramatic improvement in the quality and expectancy of life. Unfortunately, since their
discovery, antimicrobial resistance is emerged and spreading rapidly. Microbial resistance is a
big challenge to treat common infectious diseases resulting in prolonged and severe illness,
disability, and even death. (Since its discovery till now antimicrobial resistance is a huge
challenge even so) [1]. It is estimated that resistant microbes (such as bacteria, and viruses) cause
700,000 deaths each year, and these ‘superbugs’, by 2050 could cause over 10 million deaths
annually, and will cost the global economy 100 trillion USD, if present situation persist [2].

Microbes have developed several mechanisms to be drug resistant, (Several drug resistant
mechanisms in microorganisms have been identified) such as active efflux, modification of a
drug target, molecular bypass, or biofilm survival through protection provided by matrix
polysaccharides. This scenario therefore, demands the development of new approaches to combat
microbial infection. Unfortunately, discovery and antibiotic development has also slow down
over the past six decades. In contrast to the 1940s to 60s (golden age for novel antibiotics
development), no new chemical classes of broad-spectrum antibiotics are present in the drug
market may be due to limited number of potential targets available in microbes, but most
importantly due to difficulties in the identification of leads with “no or limited toxicity” in
humans [3]. Infect, it is time to rethink about the antibiotic drug development strategy before a
time came when infectious disease becomes a major cause of mortality in the world.  (before
deleterious outcome of resistant microorganisms worldwide).

One good option is to isolate therapeutically active protein having ability to interact with
polysaccharides present on surface of microorganisms and to further use them as adjuvants, by
taking advantage of their ability to overcome excessive efflux or their ability to interact with
microbe’s matrix polysaccharides. Such adjunctive therapies can be helpful in prolonging the
lives and efficiency of existing antibiotics [4]. Among different classes of proteins, plant lectins
because of their carbohydrate-binding specificity got considerable attention as antimicrobial
leads. Lectins are well known to interact with glycoconjugates present on the cell surface of the
microorganisms. The unique carbohydrate binding specificities of plant lectins can facilitate
efficacy of antimicrobial drugs by their co-administration. Hence could play a beneficial role
against microbial pathogenesis [5].

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Lectins are widely found in natural flora. A large number of lectins have been purified from
different plants, such as Araucaria angustifolia (seed), Pisum sativum (seed), Gastrodia data
(corms), Musa acuminate (fruit), Canavalia ensiformis (beans), etc. Among them, lectins from
Musa acuminate (BanLec), and Canavalia ensiformis (ConA) are focused since last two decades
mainly because of their antimicrobial potential, but till date there is no report available on the
structural studies describing their use as adjuvant antimicrobial therapy [6, 7].

Keeping in view the antimicrobial potential of plant lectins, we assume that lectins from banana
and jack beans can be explored as adjuvant in-combination with different antimicrobial drugs
and leads. Hence safe and effective options can be developed for their administration to
overcome microbial resistance. Additionally, use of X-ray crystallographic approaches will
provide more insight into lectin and ligand interactions. 

Significance of the Topic:

Antimicrobial resistance is now becoming a global healthcare challenge. Medicinal plants are
rich source of numerous therapeutic compounds such as, phytochemicals and proteins. Plant
Lectins as mentioned above are considered as antimicrobials through their carbohydrates binding
capability on microbial cell surfaces. Hence, there is an increasing demand to discover the
interaction of plant lectins with antimicrobial drugs/ leads, supporting the need of safe and
effective lectin based adjuvant therapy.

Research Question:

Do the different antimicrobial drugs and leads can have the binding capability with the plant
lectins, and hence can be co-administered with the plant lectins to minimize the onset of
antimicrobial resistance?

Hypothesis:

Being natural antimicrobial agent, lectins can be use as adjuvant with different antimicrobial
drugs and leads to enhance their antimicrobial potential.

Expected Outcomes:

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It is expected that our research will expectedly provide structural basis, to develop lectin based
alternative medicines that could even strike resistant microorganism. It can also lead to the
identification of glyco-compounds to enhance the antimicrobial activity of lectins and can
replace currently available antimicrobial treatment.

Limitations:

1) Most of the proteins are not thermally stable. It could be a limitation to strictly monitor
and maintain specific temperature throughout the purification steps.
2) Extraction buffers and their pH should be maintained properly, as small variation against
optimum conditions can lower biological activity of a protein.
3) Low yield of plant proteins is a big hurdle for their sutural studies and molecular
interactions.
4) Optimization of a particular condition to obtain well diffracting crystals is itself not an
easy task.
5) Binding of lectin with microbial agents not necessarily means inhibition of microbial
growth, as it is mentioned in the literature that some lectin shows binding but was not
able to hold microbial growth.

Objectives:

 To isolate, and purify lectins from Musa acuminate (banana), and Canavalia ensiformis
(jack beans).
 To evaluate the antimicrobial activity of isolated lectins by using a battery of
antimicrobial assays.
 To evaluate antimicrobial activity of isolated lectins in-combination with antimicrobial
drugs and leads by using antimicrobial assays.
 To crystallize purified lectins in apo-form, as well as in-complex with different
antimicrobial drugs and leads.
 To analyze the molecular interactions of selected antimicrobial drugs/ leads with purified
plant lectins by using X-ray crystallography.

Literature Survey:
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Protein-carbohydrate interactions are very crucial step in different biological processes. Lectins
are carbohydrate binding proteins with specificity in their action. Structural studies revealed that
besides their catalytic domains, these proteins also possess sugar binding domains. Sugar binding
capabilities of lectins are related to their antimicrobial activities. Lectins usually undergo
oligomerization after interaction with surface glycans of microorganisms, with this context it is
vital to understand structural basis of lectins. Carbohydrate-lectin binding takes place by
attractive forces including electrostatic, hydrogen bond and hydrophobic interactions [8, 9].

Mannose binding lectins were identified in many plant families out of which BanLec, from Musa
acuminata and Musa paradisica species, is a subgroup of Jacalin related lectin family, consisting
two subunits of 15 KDa. These subunits are unique in recognizing internal, and terminal sugar
linkages preferably glucose/mannose residues. BanLec is the only member of Jacalin related
lectins of monocot family having Beta prism type 1 fold. From therapeutic point of view,
BanLec showed antiviral activity by blocking virus entry due to high number of mannose
residues present in its glycoproteins [10, 11, 12]. Co-Crystallization of BanLec with mannose as
reported, and glucosamine, fucose, rhamanose and other sugars present on surface of
microorganism, is important for a better antimicrobial approach like glycoconjugate vaccine
development against Candida albicans, an opportunistic pathogen which possess major glycan
expressed on its cell wall i.e. phosphomannan based glycoproteins, β-glucans and chitin[13, 14,
15]. We aim to purify banana lectin from riped banana fruit, and to evaluate its therapeutic
effects vin combination with different antimicrobial drugs and leads.

Concanavalin A, lectin from Canavalia ensiformis, is a homotetramer of 26.5 KDa overall

molecular weight is 104-112 KDa. Con-A is reported to exhibit different biological activities
such as, Anti-hepatoma and anti-tumor effect against human breast cancer cells [16].
Additionally, Con-A showed binding affinity towards teichoic acid present on cell wall of
Mycobacterium species and could be further investigated for its antimicrobial activity. Keeping
in mind the importance of plant lectins as antimicrobial agent, we also aim to isolate and
crystallize Con-A from the beans of Canavalia ensiformis for detailed binding studies with
different antimicrobial drug and leads [16, 17, 18].

It is reported that lectins from plant origin showed binding with different sugars (see Table-1)
and ions i.e. calcium, magnesium, cadmium, sodium etc. However, there is no such report on
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binding of these lectins with antimicrobial drugs. Therefore, we aim to study and explore the
enhanced antimicrobial activities of different antimicrobial drugs and potential leads in
combination with plant lectins. In our proposed studies drugs-lectins interactions at molecular
level will be evaluated in depth, via X-ray crystallography, to further propose the use of lectins
as potentiator in antibiotic adjuvant therapy. On the basis of literature survey, drugs presented in
Table-2 are selected as proposed target molecules for structural studies.

Table-1: List of reported ligands/sugars that showed molecular interactions with lectins analysed
by X-ray crystallography.

Structural Studies of BanLec with different ligands PDB ID

-D-Glucose 2BNO

Methyl-3-O-beta-D-xylopyranosyl-α-D-mannopyranoside(XM) 2BMZ

Mannose 3MIU

Mannobiose 3MIU

Methyl-α-D-mannoside 1X1V

Structural Studies of Con-A with different ligands

-D-Mannose 5Z5N

Mannose 5Z5L

Trimannoside 3D4K

Methyl-α-D-glucopyranoside 1CJP
Table-2: List of proposed drugs/ligands to be analysed in this study for evaluating binding
interaction with the plant lectins.

S. No. Drugs Class

1. Fluconazole Antifungal

2. Isoniazid Antimycobacterial

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3. Pyrazinamide Antimycobacterial

4. Carbapenem Beta-lactam

5. Methicillin Beta-lactam

6. Aminosalicyclic acid Antimycobacterial

7. 5-Fluorouracil Antibacterial

8. Nitrofurantoin Antibacterial

9. Clindamycin Antibacterial

10. Chloramphenicol Antibacterial

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 Proposed Work Plan

PART-A:

By using classical techniques Evaluation of antimicrobial

lectins will be
including SDS-PAGE, size activity of isolated lectins by
isolated from banana
exclusion, and affinity using battery of antimicrobial
fruit and jack beans
chromatography techniques. assays.

PART-B:

Selection of sugars, and pure compounds (with antimicrobial activity) for co-crystallization with isolated
lectins.

PART-C:

Crystallization of
Co-crystallization in-complex Data collection, and structural
isolated lectins in
with different ligands. analysis.
apo form.

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Time Frame:
(Q = 3 months)

Time Chart
Activities
Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8

 Literature survey
 Purchasing of chemicals
 Isolation and purification of
lectin from banana and jack
beans

In-vitro antimicrobial activities

of isolated lectins with selected
drugs.

Crystallization of lectins in
apo-form as well as in-complex
with different drugs.

 X-Ray data collection and

processing (structure
solution and refinement)
 Thesis write-up, and
research publication (s).

References:
1. Jandu, J. J., Moraes Neto, R. N., Zagmignan, A., de Sousa, E. M., Brelaz-de-Castro, M.
C., dos Santos Correia, M. T., and da Silva, L. C. (2017). Targeting the immune system
with plant lectins to combat microbial infections. Frontiers in Pharmacology, 8, 671.
2. Tagliabue, A., and Rappuoli, R. (2018). Changing priorities in Vaccinology: antibiotic
resistance moving to the top. Frontiers in Immunology, 9, 1068.
3. Van Acker, H., Van Dijck, P., and Coenye, T. (2014). Molecular mechanisms of
antimicrobial tolerance and resistance in bacterial and fungal biofilms. Trends in
Microbiology, 22 (6), 326-333.
4. Gill, E. E., Franco, O. L., and Hancock, R. E. (2015). Antibiotic adjuvants: Diverse
strategies for controlling drug‐resistant pathogens. Chemical Biology & Drug Design, 85
(1), 56-78.
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5. Gavrovic-Jankulovic, M., and Prodanovic, R. (2011). Drug delivery: Plant lectins as
bioadhesive drug delivery systems. Journal of Biomaterials and Nanobiotechnology, 2
(05), 614.
6. Hamid, R., Masood, A., Wani, I. H., and Rafiq, S. (2013). Lectins: Proteins with diverse
applications. Journal of Applied Pharmaceutical Science, 3(4), S93-S103.
7. Singh, S. S., Devi, S. K., and Ng, T. B. (2014). Banana lectin: A brief review. Molecules,
19 (11), 18817-18827.
8. Sivaji, N., Suguna, K., Surolia, A., and Vijayan, M. (2019). Structural biology of plant
lectins and macromolecular crystallography in India. Current Science, 116 (9), 1490-
1505.
9. Breitenbach Barroso Coelho, L.C., Marcelino dos Santos Silva, P., Felix de Oliveira, W.,
de Moura, M.C., Viana Pontual, E., Soares Gomes, F., Guedes Paiva, P.M., Napoleão,
T.H. and dos Santos Correia, M.T., (2018). Lectins as antimicrobial agents. Journal of
Applied Microbiology, 125 (5), 1238-1252.
10. Meagher, J. L., Winter, H. C., Ezell, P., Goldstein, I. J., and Stuckey, J. A. (2005).
Crystal structure of banana lectin reveals a novel second sugar binding site.
Glycobiology, 15 (10), 1033-1042.
11. Ho, V. S., Wong, J. H., and Ng, T. B. (2007). A thaumatin-like antifungal protein from
the emperor banana. Peptides, 28 (4), 760-766.
12. Mitchell, C. A., Ramessar, K., and O'Keefe, B. R. (2017). Antiviral lectins: Selective
inhibitors of viral entry. Antiviral Research, 142, 37-54.
13. Cheung, A. H., Wong, J. H., and Ng, T. B. (2009). Musa acuminata (Del Monte banana)
lectin is a fructose-binding lectin with cytokine-inducing activity. Phytomedicine, 16 (6-
7), 594-600.
14. Colombo, C., Pitirollo, O., and Lay, L. (2018). Recent advances in the synthesis of
glycoconjugates for vaccine development. Molecules, 23 (7), 1712.
15. Cortegiani, A., Misseri, G., Fasciana, T., Giammanco, A., Giarratano, A., and
Chowdhary, A. (2018). Epidemiology, clinical characteristics, resistance, and treatment
of infections by Candida auris. Journal of Intensive Care, 6 (1), 69.
16. Adel, N., Mantawy, E. M., El-Sherbiny, D. A., and El-Demerdash, E. (2019). Iron
chelation by deferasirox confers protection against concanavalin A-induced liver fibrosis:
A mechanistic approach. Toxicology and Applied Pharmacology, 382, 114748.
17. Archibald, A. R., and Coapes, H. E. (1971). The interaction of concanavalin A with
teichoic acids and bacterial walls. Biochemical Journal, 123 (4), 665.

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 18. Wearne, K., Winter, H. C., and Goldstein, I. J. (2013). Isolation of banana lectin-A
practical scale procedure from ripe banana fruit. Preparative Biochemistry and
Biotechnology, 43 (3), 285-292.
19. Sharma, A., and Vijayan, M. (2010). Influence of glycosidic linkage on the nature of
carbohydrate binding in β-prism I fold lectins: An X-ray and molecular dynamics
investigation on banana lectin–carbohydrate complexes. Glycobiology, 21 (1), 23-33.
20. Singh, D. D., Saikrishnan, K., Kumar, P., Surolia, A., Sekar, K., & Vijayan, M. (2005).
Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable
evolutionary origin. Crystallographic and modelling studies. Glycobiology, 15 (10),
1025-1032.
21. Grunbein, M.L., Bielecki, J., Gorel, A., Stricker, M., Bean, R., Cammarata, M., Dorner,
K., Frohlich, L., Hartmann, E., Hauf, S. and Hilpert, M., (2018). Megahertz data
collection from protein microcrystals at an X-ray free-electron laser. Nature
Communications, 9 (1), 3487.
22. Chung, N. J., Park, Y. R., Lee, D. H., Oh, S. Y., Park, J. H., and Lee, S. J. (2017).
Heterometal-Coordinated Monomeric Concanavalin A at pH 7.5 from Canavalia
ensiformis. Journal of Microbiology and Biotechnology, 27 (12), 2241-2244.
23. Cianci, M., Nanao, M., and Schneider, T. R. (2019). Long-wavelength Mesh&Collect
native SAD phasing from microcrystals. Acta Crystallographica Section D: Structural
Biology, 75 (2).
24. Agrawal, B. B., and Goldstein, I. J. (1965). Specific binding of concanavalin A to cross-
linked dextran gels. Biochemical Journal, 96 (3), 23-25.
25. Doyle, R. J., Woodside, E. E., and Fischel, C. W. (1968). The concanavalin A precipitin
reaction with polyelectrolytes and polysaccharide derivatives. Biochemical Journal, 106
(1), 35.
26. Quirnheim Pais, D., Rathmann, B., Koepke, J., Tomova, C., Wurzinger, P., & Thielmann,
Y. (2017). A standardized technique for high-pressure cooling of protein crystals. Acta
Crystallographica Section D: Structural Biology, 73(12), 997-1006.

Annexure (Detailed Methodology)

1. Isolation and Purification of Plant Lectins:
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1.1. Isolation and Purification Banana lectin (ripe banana fruits)

Ripe banana fruits for this study will be purchased from local market. Following scheme will be
followed for the isolation and purification of Ban-Lac. [21-23].

Ripe banana fruits will be Pulp will then be Precipitation of

soaked in 150 mM acetic diluted and proteins will be carried
acid (overnight). homogenized for 6 out by using 65%
hours ammonium sulphate
after clarification
through cheesecloth
Affinity chromatography Dialysis of sample After centrifugation,
will be performed by against same buffer resuspension of the
using Superdex G-75 will be performed pellet will be carried
column. Elusion will be with six times out in 10 mM
carried out with 0.3 M change. phosphate-buffered
Methyl-α-D- saline solution (PBS)
Mannopyranoside. pH 7.4

Scheme-2: Protocol for the isolation and purification of the Ban-Lac.

1.2. Isolation and purification of lectin from Canavalia ensiformis (beans)

Fine powder of the Jack bean for this study will be purchased from Sigma, and following scheme
will be followed for the isolation and purification of Con-A [24, 25].

Jack bean powder + 0.5 After filtration and Resuspension of

M NaCl, incubation at 4˚ centrifugation, protein in collected precipitates in
for overnight supernatant will be deionzied water and
precipitated first with 30% dialysis with water and
and then 80% (NH4)2SO4 1M NaCl.

Affinity Affinity
Protein will be eluted by
chromatography will chromatography will
using 1M Glucose.
further be performed by be performed by using
Dialysis will be carried out
using Sephadex G-50 Superdex G-100.
against deionized water
column.

Scheme-3: Protocol for the isolation and purification of the Con-A.

2. In-vitro Antimicrobial Activity of Isolated lectins, and Lectin-Drug

Adjuvants:

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Selected drugs (presented in Table-2) in combination with isolated lectins will be evaluated for
antimicrobial activity by using battery of in-house established antimicrobial assays.

3. Crystallization of Isolated Banana Lectin In-complex with different

antimicrobial Drugs and leads:

Structural studies of isolated BanLec in-complex with different antimicrobial drugs will be
carried out by using following methodology via hanging drop vapor diffusion method [10, 19, 20].

Protein solution (5 mg/ mL) will be prepared in double distilled water containing Methyl-α-D-
Mannopyranoside (100 mM)

Samples of selected drugs will be added to protein solution, and will then be incubated for 24 h
at 298 K.

Incubated solution will be equilibrated (at 10:1 molar ratio) with crystallization buffer (0.01 M
Zinc acetate; Sodium cacodylate 0.1 M; pH 8, and 1,6 hexanediol 3 M), and will allowed to
equilibrate for a week to obtain crystals of the complexes.

Data collection and processing will be carried out by using in-house Bruker D8 Venture X-ray
diffractometer.

Finally, structure solution and refinement will be carried out by using CCP4 software
suite.

Scheme-4: Co-crystallization of Ban-Lac in-complex with different drugs, and leads.

4. Crystallization of Isolated Concanavalin A, In-complex with different

antimicrobial Drugs, and leads:

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Structural studies of isolated Concanavalin-A lectins in-complex with different antimicrobial
drugs will be carried out by using following methodology via sitting drop vapor diffusion method
[26].

Concanavalin A will be dissolved in 0.025 M HEPES pH 7.0 at a concentration of 10 mg/ml

Crystallization will be carried out by using sitting-drop method at 291 K in 200 nl drops at a
1:1 mixing ratio.

Concanavalin A will be crystallized using 6 %( w/v) PEG 8000, 0.1 M Tris pH 8.5 as the
reservoir solution.

Data collection and processing will be carried out by using in-house Bruker D8 Venture X-ray
diffractometer.

Finally, structure solution and refinement will be carried out by using CCP4 software suite.

Scheme-4: Co-crystallization of Con-A in-complex with different drugs, and leads.

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