Plant Analysis

Ganapati Ganesh Bhat

Ph.D. Scholar

P. G. Department of Studies in Biochemistry

Karnatak University

Dharwad- 580 003 (India)

Email: ********.**@*****.***

Phone: +91-974*******

Nationality: Indian

Date of Birth: 18th June 1985

Marital Status: Single

* EDUCATION:

Present Ph.D. thesis submitted to Karnatak University

Dharwad on 22nd March 2013.

(The work was carried out at P. G. Department of

Biochemistry, Karnatak University Dharwad and

Indian Institute of Science, Bengaluru,Karnataka,

India)

The study encompasses the purification,

physico-chemical characterization, crystallization

and molecular cloning of a monocot mannose binding

plant lectin from the edible epiphyte Remusatia

vivipara (RVL). This plant has got medicinal

property and is used as a folk medicine for

treating Arthritis and wound healing in certain

parts of south India. The tubers of this plant are

highly proteinaceous and are free from attack by

any insects and pests. Considering the immense

application of monocot plant lectins in the recent

past, the study was further extrapolated to exploit

the insecticidal property of Remusatia vivipara

lectin against agricultural pests of various orders

such as root-knot nematode Meloidogyne incognita,

Spodoptera litura and Helicoverpa armigera which

are the major threats for the food and commercial

crops. Further the attempt was made to understand

the mechanism of lectin action on mid gut cells of

Spodoptera demonstrated the possible involvement of

caspase-3 mediated apoptosis. Also the lectin

interacting receptors were purified and

characterized by PROTEOMICS analysis.

Based on the hypothesis of the study and the

findings the thesis is entitled as "Studies on

monocot mannose binding lectin from Remusatia

vivipara in support of its application in

agriculture"

M.Sc Biochemistry- First class (62.70 %),

Karnatak University, Dharwad. From 2005-2007

B.Sc Chemistry, Botany, Zoology- First class with DN

(78.2 %),

Dr. A. V. Baliga College of Arts and Science,

Kumta. From 2002-2005.

* Salient features of the current work and the findings:

> Recently, considering the importance and potent applications of the

monocot mannose binding lectins in medicine, agriculture and their unique

structural design, we screened several monocot plants for the presence of

lectin activity. During the course of our routine screening exercise, we

found very high hemagglutination activity in the tubers of Remusatia

vivipara, a monocot family grown in western ghat regions of India. This

plant belongs to family; Araceae of subclass monocots. R. vivipara, an

epiphyte, all of its parts including the tubers is edible and the plant

is resistant to the attack by many insect pests. Highly proteinaceous

tubers are used as folk medicine for treating inflammation, wound healing

and arthritis.

> Purification and characterization of R. vivipara lectin (RVL):

Single step affinity purification protocol was developed using

asialofetuin-Sepharose 4B as affinity matrix. RVL agglutinate rabbit

erythrocytes but not the human A, B, or O erythrocytes and its

hemagglutinating activity strongly inhibited by glycoprotein such as,

mucin, asialomucin, asialofetuin and thyroglobulin. The lectin activity

is stable at temperatures up to 80 0C and under wide pH range of 2.0 to

9.0.

Purified lectin was homogeneous as determined by SDS-PAGE and gel

filtration chromatography with homo tetramer molecular mass of 49.50kDa.

But the mass determined by MALDI showed two distinct sub units of 12.0

kDa and 12.7 kDa suggesting the cleaved products of subunit polypeptide,

commonly seen in monocot MBL's, this was confirmed from dynamic light

scattering studies, N- terminal analysis and by molecular cloning of the

lectin coding gene. Purified lectin was successfully crystallized and

later the X-ray crystallographic structure determined at 2.1

resolution, in collaboration confirmed the cleavage of the precursor poly

peptide chain resulting to yield hetero-dimer. These studies demonstrated

that RVL is a dimer with Mr 49.5, derived from single polypeptide chain

of 233 amino acids yielding two subunits because of cleavage between 116

and 117 residues. These findings also indicated the presence of two

putative domains in RVL, containing one mannose binding site per domain.

> Cloning of full length cDNA of RVL and sequence analysis

The full length lectin gene was isolated and cloned into T/A cloning

vector to deduce the amino acid sequence of the protein. The sequence

confirmed the earlier findings, presence of two different polypeptides in

the lectin and the multiple sequence analysis has shown that the lectin

is undergoing cleavage as a post translational modification. These

observations also indicated the presence of two putative domains in RVL,

containing one mannose binding site per domain.

> Entomotoxic properties of purified RVL:

Considering the entomotoxic properties of mannose binding plant lectins

the present study was extrapolated to evaluate the toxicity of RVL on

the agricultural pests of different orders such as root-knot nematode

Meloidogyne incognita, cotton leaf worm Spodoptera litura, and boll worm

Helicoverpa armigera. These insects are polyphagous and are the major

threat for many of the agricultural food crops and crops of economic

importance and are responsible for overall loss of 25-30% agricultural

production all over the world.

A. Toxicity of RVL on Meloidogyne incognita:

The purified RVL exerts concentration dependent toxicity on

Meloidogyne incognita, a root knot nematode. Dose dependent bioassays

conducted with M. incognita showed highest mortality of 88% at lectin

concentration at 30 g/ml in 48 hrs and as high as 50% mortality even at

7.5 g/ml lectin concentration indicating the potential nematicidal

activity of the lectin. Fluorescent confocal microscopic studies

demonstrated the binding of RVL to specific regions of the alimentary-

tract and exhibited potent toxic effect on M. incognita. RVL-mucin

complex failed to interact with the gut confirming the receptor mediated

lectin interaction. The above results suggested that RVL can be very

good molecule for its exploitation in agriculture, for the development

of nematode resistant transgenic-crops.

B. Toxicity of RVL on Spodoptera litura:

S. litura commonly known as Cotton leaf worm is a lepidopteran pest

of cutting and chewing type causing serious damage to several important

crops including vegetables. Bioassays were carried out by feeding

artificial diet with different concentrations of purified lectin on 2nd

instar larvae. The effect of RVL on the growth, development and survival

of the larvae was recorded during the experiment. The larvae were fed

on the artificial diet supplemented with purified lectin (RVL) at

different concentrations (0.00; 0.020; 0.040 &0.060%), showed remarkable

larval mortality and serious impairment in development as compared to

control group. The mortality was directly proportional to the lectin

concentration. The highest mortality of 57.41 8.48 % was observed at

highest concentration of 0.06% tested with toxicity value (LC50) of

510 g/g of artificial diet.

In order to understand the mechanism of lectin action,

interaction of lectin with mid-gut epithelium was investigated by immuno-

histochemical and western blot analysis. These experiments revealed the

interaction of RVL with mid-gut is by a specific glycan-mediated

process. Further the immuno-histochemical experiments to determine the

cytoplasmic and nuclear variation upon exposure to RVL has provided a

hint for the occurrence of apoptosis in the insect gut. Further studies

on caspase-3 expression in the cytoplasm and DNA fragmentation studies

of RVL treated mid-gut epithelial cells have strongly supported the

earlier observation of caspase dependent apoptosis. Two RVL binding

receptors with mass 66 and 39 kDa were identified from the gut

epithelial cells of S. litura larvae by western blot studies.

C. Effect of RVL on the cotton bollworm H. armigera.

H. Armigera commonly known as cotton boll worm is also a lepidopteran

pest similar to S. litura causing economic losses to agriculture crops.

Bioassays were carried out on the artificial diet supplemented with

purified RVL at different concentrations such as 0.02%, 0.04%, and 0.06%

to determine the effect on the larvae of H. armigera. Unlike the effect

on S. litura, RVL exert strong anti-feeding effect in the early instars

larvae resulting in remarkable death rate. The highest mortality of

81.48% was observed at a concentration of 0.06% of RVL, in the early 3rd

instar stage of the larvae. Subsequently the mortality of 48.14% and

29.62 % was observed at concentrations of 0.04% and 0.02% respectively

indicating the effectiveness induced by RVL. Surprisingly when the

larvae which were reluctant to feed on diet supplemented with RVL, fed

on the normal control diet without lectin, they fed well and grown

equally well as control groups. But none of the moths survived more than

3 days after emergence. The above results demonstrated the effect

induced by RVL on H. armigera is reversible.

> Carbohydrate specificity of RVL by glycan array analysis and

characterization of lectin receptors on the mid gut of Spodoptera

litura.

A. Determination of fine sugar specificity of RVL by glycan array

analysis:

Apart from determining the sugar binding properties by hapten

inhibition studies, the fine sugar specificity of RVL was determined at

the consortium for functional glycomics (CFG) by using biotinylated

lectin and the array consisted of 511 different glycans. The glycans

presented on the array slide were either naturally occurring or

synthetic ones of wide ranging structures which are part of

physiologically important animal glycoproteins. Fine sugar specificity

analysis (www.functionalglycomics.org RVL_v4.2_2138) revealed that RVL

recognizes only N-linked complex glycans but not the O- linked sugar

chain also none of the simple monosaccharides or their derivatives.

These results demonstrated that tri-mannosyl pentasacharide core is an

absolute necessary for RVL binding to N-linked complex glycans and it

showed greater affinity towards the complex fucosylated core structures,

however fucosylation is not mandatory for sugar binding. The glycan

array analysis also showed the impressive binding of RVL to some glycans

of pathological significance, for eg; a fucosylated high mannose N-

glycan associated with hepatic disorders like cirrhosis and

hepatocellular carcinoma. RVL also bind to Man?1-6(Man?1-3) Man?1-

6(Man?1-2Man?1-3) Man?1-4GlcNAc?1-4GlcNAc?-Sp12 which is a part of CA-

125 (MUC 16), a well known tumor marker most widely used for the

diagnosis of human epithelial ovarian cancer and also for other cancers.

Most important and significant revelation made by RVL glycan array

analysis is the binding to glycan; Man?1-6(Man?1-3) Man?1-6(Man?1-3)

Man?1-4GlcNAc?1-4GlcNAc?-Sp12, which is part of HIV envelope

glycoprotein GP120 involved in HIV infection through the interaction

with CD4 cells (Scanlan, C.N., et al., 2007).

Hence RVL, a monocot mannose binding lectin can have potential bio-

medical applications particularly in cancer biology and virology.

However the glycan array analysis provided insights into the

contribution of not only the individual sugar moieties but also the type

of linkage between them on the binding affinities.

B. Characterization of the lectin receptors from the mid gut epithelial

cells of Spodoptera litura

Glycan array analysis data provided valuable information on RVL's

carbohydrate recognition property, which lead to understand the nature

of the receptors on the insect epithelial gut cells interacting with the

lectin. The possible receptors involved in the RVL recognition on the

mid-gut of S. litura larvae were characterized by ESI-Q-TOF analysis.

The study consisted of purification of membrane proteins

from the mid gut epithelial cells of S. litura by affinity

chromatography on RVL-sepharose column. Affinity eluted protein

receptors separated by SDS-PAGE were analyzed and characterized by ESI-Q-

TOF. SDS-PAGE analysis indicated the presence of three receptors

recognized by the RVL, with Mr of 14.3, 39.0 and 66.0 respectively.

Further, ESI-Q-TOF analysis was carried out to characterize the possible

receptors recognized by RVL. Total of 3235 peptides were identified in

the tryptic digest of affinity eluted membrane protein fraction from the

ESI-spectra. The data was analyzed using NCBI non redundant and

Swissprot databases by selecting the taxonomy of Drosophila melanogastor

since it is considered as model organism.

Demonstration of the toxic effect of RVL on Meloidogyne

incognita, Spodoptera litura and Helicoverpa armigera, known for the

heavy crop damages, opens new avenues to develop transgenic crops for

resistance against these pests. Since the full length gene encoding the

lectin has been cloned, it is hoped to clone and express the lectin gene

in plant system by developing suitable protocols. Preliminary findings

indicating the caspase mediated apoptotic effect of RVL on mid gut

epithelial cells of S. litura larvae leading to mortality would address

the biosafety concerns of the lectin in transgenic plants.

* TECHNICAL EXPERTISE:

> Protein purification and analysis: Protein purification from fungal and

plant sources including bacteria and well acquainted with the use of ACTA

Prime Protein purification system, Ion exchange, Affinity and Gel

filtration chromatography. Resolution of protein samples on SDS and

native PAGE and their visualization by comassie staining, silver staining

and western blotting. Isolation of membrane proteins from cell surfaces.

Colorimetric and spectrophotometric estimation of proteins. Isolation of

isozymes from fungi and analysis.

> Biochemical assays: Hemagglutination and hapten inhibition assays for

lectin activity/sugar specificity.

> Physicochemical characterization techniques: Acquainted with Dynamic

Light Scattering, Circular Dichroism, Preparation of sample for N-

terminal sequencing, MALDI-TOF, and setting for crystallization of

protein.

> Molecular biology techniques: Hands on experience in plasmid isolation,

isolation of DNA from bacteria, plant and fungi; agarose gel

electrophoresis, Restriction analysis, PCR analysis, Ligation and

Transformation, expression and purification of recombinant protein from E

.coli. The sequencing and analysis of the gene, using automated DNA

sequencer.

> Cell biology techniques: Acquaintance in maintaining adhering type of

cancer cells (established) such as PA-1 and HT29 cells; Cell counting,

proliferation and antiproliferation studies; Dose and time dependant cyto-

toxicity studies. Western blotting studies to understand the mechanism of

lectin action. Acquaintance in isolating the stem-cells from insect mid-

gut.

> Microscopic studies: Acquainted with Fluorescence microscopic studies and

confocal microscopy.

> Microbiological: Basic microbiological techniques, isolation of pure

culture and maintenance.

> Bioinformatics techniques: Sequence analysis of protein and nucleic acids

and primer designing.

> Membrane protein purification and Proteomic analysis: Purification of

membrane protein using lectin affinity chromatography; Tryptic digestion

and preparation of the protein sample for ESI-Q-TOF analysis. Protein

analysis and identification using proteomic softwares and databases.

> Immuno-histochemical technique: Acquainted with dissection of insects,

gut isolation; Tissue embedding, microtome sectioning and processing and

analysis.

> Bioassay of lectins: Standardization of assay condition, artificial diet

and study of life cycle of insect, bioassay of lectins supplemented in

artificial diet and transgenic leaves.

* WORK EXPERIENCE :

> Worked as Technical Assistant from 1st August 2007 to 24th October 2008

at Institute of Agricultural Biotechnology (IABT), University of

Agricultural Sciences, Dharwad-580005.

> Worked as a Trainee from 1st April 2008 to 30th April 2008 at Molecular

Biophysics Unit, Indian Institute of Science, Bengaluru, Karnataka, India

> Trained in automated DNA sequencing, using automated DNA sequencer by LAB

India (Applied Biosystems), Gurgaon, Haryana, India

> Acquainted in membrane protein purification, preparation of sample for

ESI-Q-TOF and analysis using the proteomic technique and softwares at

Agilent Technologies, Bangalore, India.

* AWARDS AND FELLOWSHIPS:

. Awarded the Junior Research Fellowship under the University Grant

Commission (UGC) - Research Fellowship in Sciences for Meritorious

Student (RFSMS) from 2010- 2011 at P. G. Dept. of Biochemistry, Karnatak

University, Dharwad, Karnataka, India.

. Awarded the Senior Research Fellowship under Council for Scientific and

Industrial Research (CSIR) funded project from 2011- till date at P. G.

Dept. of Biochemistry, Karnatak University, Dharwad, Karnataka, India.

* POSTER PRESENTED:

Presented a poster at 32nd Annual Convention of Indian Association for

Cancer Research & International Symposium: Infection & Cancer, organized

by Dr. B.R. Ambedkar Center for Biomedical Research (ACBR) and University

of Delhi, from 13th -16th Feb. 2013, titled "Sclerotium rolfsii lectin

induces stronger inhibition of growth in human breast cancer cells than

in normal mammary epithelial cells by induction of cell apoptosis".

* CONFERENCE ATTENDED:

Attended, National Conference on Snakebite Management; 2nd Annual

Conference of Toxicological Society of India, held at Mysore, Karnataka,

India from December 10 to 12, 2012.

* WORKSHOP ATTENDED:

Attended interactive international workshop on Thrombosis and

Haemostasis: Discovery and Development of Tools and Therapeutics, from

December 8-9, 2012

* RESEARCH PUBLICATIONS:

1. Ganapati G. Bhat, Kartika N. Shetty, Nagaraja N. Nagre, Vivek V.

Neekhra,

S. Lingaraju, Ramesh S. Bhat, Shashikala R. Inamdar, K. Suguna, Bale M.

Swamy

"Purification, characterization and molecular cloning of a monocot

mannose-binding lectin from Remusatia vivipara with nematicidal

activity."

Glycoconjugate Journal (2010) 27: 309-320.

2. Kartika N Shetty, Ganapati G Bhat, Shashikala R Inamdar, Bale M Swamy

and K Suguna. "Crystal structure of a ?-prism II lectin from Remusatia

vivipara".

Glycoibology (2012) 22: 56-69.

3. V. Neekhra, Ganapati G. Bhat, Y. S. Bhagat, S. Lingaraju, R. S. Bhat,

Nematicidal activity of Remusatia vivipara lectin expressed in

Escherichia coli, Current Science (2011) 101(2): 150-151.

* PATENT:

1. Indian patent filed on 19th JAN 2009 (Application Number 122/CHE/2009 A)

Entitled

"PLANT TUBER LECTINS AND THEIR USE IN NEMATODE CONTROL" in collaboration

with University of Agricultural Sciences, Dharwad. Inventors: Ramesh

Bhat, Vivek Neekhra, S. B. Mallesh, S. Lingaraju, Ganapati Bhat,

Suneelkumar Basingi, B.M. Swamy, M.S. Kuruvinashetti.

* NUCLEOTIDE SEQUENCE DEPOSITED:

1. Vivek Neekhra, Ramesh Bhat, Chandrashekhar T.M., Ganapati Bhat,

Suneelkumar Basingi, S. S. Udikeri, B.M. Swamy, M. S. Kuruvinashetti,

Nucleotide sequence of novel lectin gene (RVL) deposited in international

nucleotide database, National Centre for Biotechnology and Informatics

(NCBI), Accession number EU924066, 2007.

* REFERENCES:

1. Dr. B.M. Swamy (Research Supervisor)

Professor of Biochemistry

Dept. of Biochemistry

Karnatak University

Dharwad- 580 003

Karnataka, INDIA

Phone: +91-836-*******, +91-836-*******

Email: ********@*****.**.**

2. Dr. Shashikala R. Inamdar

Professor of Biochemistry

Dept. of Biochemistry

Karnatak University

Dharwad- 580 003

Karnataka, INDIA

Phone: +91-836-*******, +91-836-*******

Email: ************@*****.**.**

3 Dr. K. Suguna

Scientist

Molecular Biophysics Unit

Indian Institute of Science

Bangalore- 560 012

Karnataka, India

Phone: +91-80-229*****

Email : *******@***.****.*****.**

DECLARATION

I hereby declare that the above information given by me is true to the best

of my knowledge & belief.

Place: Dharwad

Ganapati Bhat

CURRICULUM VITAE

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