1. ir. Mohamad HAMSHOU
Thesis submitted in fulfillment of the requirements for the degree of
Doctor (PhD) in Applied Biological Sciences
Toxicity and mode of action of fungal lectins
in pest insects important in agriculture
2.
3. Promoters: Prof. dr. ir. Guy Smagghe
Ghent University
Department of Crop Protection
Laboratory of Agrozoology
Prof. dr. Els J.M. Van Damme
Ghent University
Department of Molecular Biotechnology
Laboratory of Biochemistry and Glycobiology
Dean: Prof. dr. ir. Guido Van Huylenbroeck
Rector: Prof. dr. Paul Van Cauwenberge
4.
5. Mohamad Hamshou (2012). Toxicity and mode of action of fungal lectins in pest insects
important in agriculture. PhD thesis, Ghent University, Ghent, Belgium.
ISBN-number 978-90-5989-525-6
The author and the promoters give the authorization to consult and to copy parts of this work
for personal use only. Any other use is limited by the Laws of Copyright. Permission to
reproduce any material contained in this work should be obtained from the author.
The promoters: The author:
Prof. dr. ir. Guy Smagghe Prof. dr. Els JM Van Damme ir. Mohamad Hamshou
6.
7. Members of the examination committee
Prof. dr. ir. Guy Smagghe (promoter)
Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. Els JM Van Damme (promoter)
Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University,
Belgium
Prof. dr. ir. Patrick Van Damme (chairman)
Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. ir. Monica Höfte
Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. ir. Marie-Christine Van Labeke
Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. Jozef Vanden Broeck
Department of Biology, Animal Physiology and Neurobiology, Zoological Institute, Katholieke
Universiteit Leuven
Prof. dr. ir. Peter Bossier
Department of Animal Production, Laboratory of Aquaculture & Artemia Reference Center,
Faculty of Bioscience Engineering, Ghent University, Belgium
8. ACKNOWLEDGMENT
I acknowledge the presence of God who created me and gave me this rare privilege to
achieve my dream of attaining the highest qualification. This thesis is an output of several
years of research that has been done since I came to Ghent. Since that time, I have worked
with many people whose support and collaboration in various and diverse ways contributed
to this great success of my thesis. It is a pleasure to convey my gratitude to them all in my
humble acknowledgment.
I am highly indebted to my supervisors Prof. Dr. ir. Guy Smagghe and Prof. Dr. Els Van
Damme who taught and supervised me during these years of unraveling the mysteries behind
lectin-insect interactions in the Laboratory of Agrozoology and the Laboratory of
Biochemistry and Glycobiology. Guy and Els, it is a great honor to work with you. Without
any doubt, your efforts were putting me on the right path. I will never forget your guidance
and the help you gave me even during weekends, holidays and all other opportunities “Heel
Hartelijk Bedankt”.
My kind regards to Prof. Dr ir Hussein AL-Mohammad (Aleppo University) and Prof. Dr ir.
Roland Verhé who first introduced me to UGent and helped me to find the opportunity to do
my PhD in UGent.
I sincerely thank the chairman of the jury committee, Prof. Dr. ir. Patrick Van Damme and
the other jury members, Prof. Dr. ir. Monica Höfte, Prof. Dr. ir. Marie-Christine Van
Labeke, Prof. Dr. Jozef Vanden Broeck, Prof. Dr. ir. Peter Bossier.
I wish to express my profound appreciation to my colleagues at the laboratory of
Agrozoology for the friendly atmosphere and cooperation, Prof. dr. ir. L. Tirry, Prof. dr. ir. P.
De Clercq, S. Shahidi-Noghabi, A. Sadeghi, A. Jalali, E. De Geyter, S. Bahrami-Kamangar,
J. Maharramov, S. Jacques, O. Christiaens, R. De Wilde, T. Walski, Na Yu, N. De Zutter, A.
Billiet, N. Shoker, N. Berkvens, M. Bonte, T. Soin, Yves Verhaegen, Katrien Michiels, B.
Ingels, P. Demaegt, T. Machtelinckx, H. Huvenne, J. Bonte, A. Bryon, W. Dermauw, T. Van
Leeuwen, S. Maes, J. Moens, G. Herregods, J. Liu, K. Maebe, D. Staljanssens, P. Van
Nieuwenhuyse, I. Meeus, H. Mosallanejad and S. Caccia.
I express my deep gratitude to every member of the Departement of Molecular
Biotechnology, N. Lannoo, E. Fouquaert, G. Vandenborre, B. Al Atalah, A. Delporte, Ch.
Shang, K. Stefanowicz, D. Schouppe, B. Nagels, J. Van Hove and W. De Vos.
9. I must thank D. Van De Velde, R. Van Caenegem, L. Dierick, B. Vandekerkhove, R.
Termote-Verhalle, K. Plas, I. Tilmant, G. Meesen, S. De Schynkel and F. De Block, and the
technical and assistant staffs of the laboratories.
Hereby I also thank Prof. Dr. Kris Gevaert and Bart Ghesquière (VIB, Department of
Medical Protein Research) for their help with the proteomics analysis.
I sincerely thank Ruben De Wilde for his kind help of Dutch translation of the thesis
summary.
I would like to express my gratitude to all my Syrian friends in Belgium and their families
who have helped me during my study, especially Tarad, Abd Al Karim, M. Khlosy, M. AL-
Abed, Tamer, Kosy, M. Shehab, M. Moslet, M. Al- Shoker, M. Akash, M. AL-Hazaa, Hanan,
Fateh, Raki, Ammar, Ehab, Ola and many other friends of the Syrian community in Ghent.
I also wish to send my sincere gratitude to the General Commission for Scientific
Agricultural Research and the Ministry of Higher Education (especially Mrs. Eyman & Heba)
in Syria who supported me to pursue my stay and education in Belgium. I would like also to
send my gratitude to the Syrian embassy in Belgium (especially Mr. Yamen & Fayez).
I am very grateful to my mother and father. Their prayers, passionate encouragements and
generosities have followed me everywhere to give me a lot of power. My deepest gratitude
goes to my sisters and brothers. I wish to send my best regards to my wife’s family especially
my mother and father-in-low. I wish all of you a prosperous life full of happiness and health.
My lovely wife “Dalal” and my adorable children “Maria, Ahmad and Wesam”, you were
the main supporters of me along my entire PhD thesis. I am deeply grateful for your patience
and sacrifices. I hope I can compensate you with all my love for all the moments which I
spent far away from you.
Mohamad
May 2012
10. Table of content
List of abbreviations
Scope
Chapter 1: literature
1.1 AGRICULTURE 2
1.2 INSECTS 2
1.2.1 Hemiptera 4
1.2.2 Lepidoptera 6
1.2.3 Insect gut 8
1.2.4 Insect cell lines 10
1.3 CROP PROTECTION 11
1.3.1 Current control strategies 12
1.3.1.1 Lectins as bio insecticidal agent 12
1.4 INSECT GLYCOSYLATION PATTERNS 15
1.5 APOPTOSIS 17
1.5.1 The insect caspases 20
1.6 FUNGAL LECTINS: their toxicity and antiproliferative activity 24
1.6.1 Basidiomycota 30
1.6.2 Ascomycota 42
1.6.3 Discussion 45
1.6.3.1 Classification 45
1.6.3.2 Localization 47
1.6.3.3 Specificity 48
1.6.3.4 Molecular mass and subunit composition 48
1.6.3.5 Biological activity 48
1.6.3.5.1 Anti-virus activity 48
1.6.3.5.2 Anti-fungal activity 49
1.6.3.5.3 Anti-amoeba activity 49
1.6.3.5.4 Anti-nematode activity 49
1.6.3.5.5 Anti-insect activity 50
11. 1.6.3.5.6 Anti-mice/rat activity 50
1.6.3.5.7 Cytotoxicity and antiproliferative activity 50
1.6.3.6 Mechanisms of fungal lectin activity 51
1.6.4 Conclusions 51
Chapter 2: Analysis of lectin concentrations in different Rhizoctonia solani strains
2.1 ABSTRACT 54
2.2 INTRODUCTION
…………………………………………………………….…………….
55
2.3 MATERIALS AND METHODS 57
2.3.1 Isolates and growth conditions 57
2.3.2 Protein extraction 57
2.3.3 Determination of total protein content 58
2.3.4 Analysis of lectin activity in different Rhizoctonia strains 58
2.3.5 Gel electrophoresis 58
2.4 RESULTS 58
2.4.1 Agglutination assays 58
2.4.2 Protein analysis 59
2.5 DISCUSSION 60
Chapter 3: Entomotoxic effects of fungal lectin from Rhizoctonia solani
towards Spodoptera littoralis
3.1 ABSTRACT 64
3.2 INTRODUCTION
…………………………………………………………….…………….
65
3.3 MATERIALS AND METHODS 66
3.3.1 Isolation of RSA 66
3.3.2 Insects 67
3.3.3 Effects of RSA feeding on insect survival, growth and development 67
3.3.4 Effect of RSA combined with Bt toxin 68
3.3.5 Statistical analysis 68
3.4 RESULTS 69
3.4.1 Effects of RSA feeding on insect survival, growth and development 69
3.4.2 Effects of RSA combined with Bt toxin 73
3.5 DISCUSSION 73
12. Chapter 4: Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its
interaction with insect tissues and cells
4.1 ABSTRACT 78
4.2 INTRODUCTION
…………………………………………………………….…………….
79
4.3 MATERIALS AND METHODS 81
4.3.1 Pea aphids 81
4.3.2 Insect midgut CF-203 cell line and culture conditions 81
4.3.3 Purification of SSA 81
4.3.4 FITC-labeling of SSA 81
4.3.5 Treatment of A. pisum with SSA via artificial liquid diet 82
4.3.6 Histofluorescence for localization of SSA in aphid body tissues 83
4.3.7 Cytotoxic effect of SSA in insect midgut CF-203 cells 83
4.3.8 DNA fragmentation analysis 84
4.3.9 Caspase-3 activity assay 84
4.3.10 Uptake of SSA in midgut CF-203 cells 84
4.3.11 Effect of saponin on toxicity and uptake of SSA in midgut CF-203 cells 85
4.3.12
Effect of carbohydrates and glycoprotein on toxicity of SSA in midgut
CF-203 cells
85
4.4 RESULTS 86
4.4.1 Insecticidal effects of SSA on pea aphids 86
4.4.2 Localization of SSA upon feeding in aphid body tissues 86
4.4.3 Cytotoxicity of SSA in insect midgut CF-203 cells 87
4.4.4
DNA fragmentation and caspase-3 activity in midgut CF-203 cells upon
exposure to SSA
87
4.4.5 Internalization of SSA in midgut CF-203 cells 89
4.4.6
Inhibitory effect of carbohydrates and glycoprotein on SSA toxicity in
midgut CF-203 cells
90
4.5 DISCUSSION 91
Chapter 5: High entomotoxic activity of the GalNAc/Gal-specific Rhizoctonia solani
lectin in pest insects relies on caspase 3-independent midgut cell apoptosis
5.1 ABSTRACT 100
5.2 INTRODUCTION
…………………………………………………………….…………….
101
5.3 MATERIALS AND METHODS 102
13. 5.3.1 Insects 102
5.3.2 Purification of RSA and labeling with FITC 102
5.3.3 Treatment of S. littoralis with RSA via artificial diet 102
5.3.4 Treatment of A. pisum with RSA via artificial diet 103
5.3.5 Histofluorescence procedures 103
5.3.6 Bioassay with insect midgut cell cultures 103
5.3.7 Effect of sugars on cell toxicity of RSA in midgut CF-203 cells 103
5.3.8 Uptake of RSA in CF-203 cells 103
5.3.9 Primary cell cultures 104
5.3.10 Effect of saponin on the cytotoxicity and uptake of RSA in CF-203 cells 104
5.3.11 DNA fragmentation and nuclear staining with Hoechst in the midgut cells 104
5.3.12 Caspase activity assay in midgut cells 105
5.3.13 Isolation of binding partners of RSA from the membrane of midgut cells 105
5.4 RESULTS 107
5.4.1 Insecticidal effects of RSA on cotton leafworm caterpillars and pea aphids 107
5.4.2 Localization of RSA in the insect body of caterpillars and aphids 107
5.4.3 Cellular toxicity of RSA in midgut cells 110
5.4.4 Effect of carbohydrates on RSA toxicity in midgut CF-203 cells 111
5.4.5 Uptake of RSA in the midgut cells 111
5.4.6
DNA fragmentation analysis and nuclear condensation in midgut cells by
RSA
113
5.4.7 Caspase activity in midgut cells upon exposure to RSA 114
5.4.8
Proteomic analysis of soluble and membrane proteins of midgut cells bound
to RSA column
115
5.5 DISCUSSION 118
Chapter 6: GalNAc/Gal-binding Rhizoctonia solani agglutinin has antiproliferative
activity in Drosophila melanogaster S2 cells via MAPK and JAK/STAT signaling
pathways
6.1 ABSTRACT 124
6.2 INTRODUCTION
…………………………………………………………….…………….
125
6.3 MATERIALS AND METHODS 126
6.3.1 Isolation of lectins and labeling with FITC 126
6.3.2 Cell proliferation assay 127
6.3.3 Effect of carbohydrates on RSA antiproliferative activity on S2 cells 128
6.3.4 RSA activity in S2 cells following pre-incubation with kinase inhibitors 128
14. 6.3.5 Internalization assay 128
6.3.6 DNA fragmentation analysis in S2 cells 128
6.3.7 Nuclear staining with Hoechst dyes 129
6.3.8 Proteomic analysis of the RSA binding proteins in the membrane of S2 cells 129
6.4 RESULTS 131
6.4.1 RSA causes inhibition of cell proliferation in S2 cells 131
6.4.2 Importance of carbohydrate binding for antiproliferative activity of RSA 131
6.4.3 Binding and internalization of RSA compared to plant lectins 133
6.4.4 RSA treatment does not induce apoptosis 134
6.4.5 Effect of kinase inhibitors on RSA activity 134
6.4.6
Proteomic analysis of membrane proteins of S2 cells retained on RSA
affinity column
135
6.5 DISCUSSION 136
Chapter 7: GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES FOR
FUTURE RESEARCH
7.1 GENERAL DISCUSSION 146
7.1.1 Fungi as a source for bioactive compound 146
7.1.2 Fungal lectins as bio-insecticidal proteins 146
7.1.3 The midgut as primary target for RSA and SSA 149
7.1.4 Study of RSA and SSA binding at cellular level 150
7.1.5 Investigation of the mode of action of RSA and SSA at cellular level 151
7.1 GENERAL CONCLUSIONS 156
7.2 PERSPECTIVES FOR FUTURE RESEARCH 158
Summary/Samenvatting 161
Summary 162
Samenvatting 165
References 169
Curriculum Vitae 205
Appendix 211
15. List of abbreviations
AAL Agaricus arvensis lectin
ABL Agaricus bisporus lectin
ACL Agrocybe cylindracea lectin
AG Anastomosis group
ALG-2 Apoptosis-linked gene-2
ANOVA Analysis of variance
APA Allium porrum agglutinin
ASAL Allium sativum leaf agglutinin
BEL Boletus edulis lectin
Bm5 Ovarian insect cells
BPA Bauhinia purpurea agglutinin
Bt Bacillus thuringiensis
BVL Boletus venenatus lectin
CARD Caspase recruitment domain
CF-203 Midgut insect cells
CGL2 Coprinopsis cinerea galectin
CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate
CL Confidence limits
CNL Clitocybe nebularis lectin
ConA Canavalia ensiformis agglutinin
CPB Fat body insect cells
cry Crystal toxin of Bacillus thuringiensis
Cut Outer cuticle
DAP 1,3-diaminopropane
DED Death effector domain
DISC Death-inducing signal complex
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
16. FADD Fas-associated death domain
FAF-1 Fas-associated protein factor-1
Fas Death receptor on the cell surface
FBS Fetal bovine serum
FDR false discovery rate
FIP Fungal Immunomodulatory Protein
FITC Fluoresceine isothiocyanate
FVL Flammulina velutipes lectin
Gal Galactose
GalNAc N-acetylgalactosamine
GCL Ganoderma capense lectin
GFL Grifola frondosa lectin
GLL Ganoderma lucidum lectin
GNA Galanthus nivalis agglutinin
GPCR G-protein-coupled receptor
H2O2 Hydrogen peroxide
HEA Hericium erinaceum lectin
HEPES
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-
Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)
HIV-1 Human Immunodeficiency Virus 1
HPLC High performance liquid chromatography
IC50 The half maximal inhibitory concentration
IPM Integrated Pest Management
IRA Iris hybrid agglutinin
IUL Inocybe umbrinella lectin
JAK Janus kinase
KDa Kilodalton
KL-15 Boletopsis leucomelas lectin
LC50 The median lethal dose
LD50 The median lethal dose
LT50 Median lethal time
Lum Insect gut lumen
MEK MAP kinase
17. MG Midgut
MIC Minimum Inhibitory Concentration
mM Millimolar
MOA Marasmius oreades lectin
MTT (3-(4,5)dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PAL Pholiota adiposa lectin
PBS Phosphate buffered saline
PCD Programmed Cell Death
PCL Pleurotus citrinopileatus lectin
PeCL Penicillium chrysogenum lectin
PHA Phaseolus vulgaris agglutinin
PJL Paecilomyces japonica lectin
PM Peritrophic membrane
PMSF Phenylmethylsulphonyl fluoride
PNA Peanut agglutinin
POL Pleurotus ostreatus lectin
RBL Rhizoctonia bataticola lectin
RDL Russula delica lectin
RFU Relative fluorescence units
RLL Russula lepida lectin
RLU Relative luminescence units
RSA Rhizoctonia solani agglutinin
RTK Receptor tyrosine kinases
S2 Embryonic insect cells
SCL Schizophyllum commune lectin
SD Standard error
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SEM Standard Error of the Means
SNA-I’ Sambucus nigra agglutinin I’
SNA-II Sambucus nigra agglutinin II
SPSS
Statistical Product and Service Solutions (formerly Statistical
Package for the Social Sciences)
SRL Sclerotium rolfsii lectin
SSA Sclerotinia sclerotiorum agglutinin
19. Scope
Several plant lectins have been reported to possess insecticidal activity towards different pest
insects. However, until now very little is known about the insecticidal activity of fungal
lectins. Therefore the main aim of this PhD research was to investigate the insecticidal
activity of some fungal lectins and to study their mode of action.
Chapter 1 gives a literature review about insects and lectins. The first part presents a survey
on the control of pest insects, the insect midgut, glycosylation in insects, regulation of cell
death in insects and the pest insects used in this project. In the second part of this chapter, an
overview is presented on fungal lectins with emphasis on the toxicity and antiproliferative
activity of these lectins towards different organisms.
The first aim of this work was to find a Rhizoctonia solani strain(s) that expresses a high
concentration of Rhizoctonia solani agglutinin (RSA) which would allow to purify sufficient
amounts of RSA for (bio)assays with insects and insect cells. In chapter 2 ten R. solani
strains belonging to different anastomosis groups were screened for the presence and the
amount of RSA in their mycelium as well as in the sclerotia. The major aim was to identify a
Rhizoctonia strain expressing high levels of lectin.
The second aim was to determine the insecticidal activity of RSA. In chapter 3 the effects of
RSA on the growth, development and survival of an economically important caterpillar in
agriculture and horticulture, the cotton leafworm, Spodoptera littoralis were investigated by
rearing this insect on an artificial diet containing different concentrations of RSA.
The third aim of this project was to study the insecticidal activity and the mode of action of
the fungal lectin isolated from Sclerotinia sclerotiorum (SSA). In chapter 4 the activity of
SSA on the survival of the piercing-sucking pest insect pea aphid Acyrthosiphon pisum was
studied using a liquid artificial diet. Moreover, binding of SSA to different tissues in the pea
aphid body was investigated upon oral exposure to FITC-labeled SSA. Further assays were
done at the cellular level using the insect midgut cell line (CF-203) to answer several
questions related to the toxicity of SSA. Answering these questions will help to understand
the mechanism of action of SSA.
The fourth aim was to investigate the mode of action of RSA. Since some proteins are active
against Lepidopteran insects but not to Hemipterans RSA was also tested for toxicity against
the pea aphid, A. pisum in chapter 5. Moreover, the target sites for RSA in the pea aphid as
well as in the cotton leafworm were analyzed using FITC-labeled RSA. To better understand
20. the mode of action of RSA, in vitro experiments were done using midgut CF-203 cells. First
the activity and interaction of RSA with CF-203 cells were investigated. Several experiments
were performed to examine the dependency of RSA activity on apoptosis induction including
DNA fragmentation, nuclear condensation and caspase activation. Second, RSA affinity
chromatography of soluble and membrane extracts of CF-203 cells was performed to identify
putative glycosylated proteins as potential binding partners for RSA.
To determine whether or not the activity of RSA is cell or organism dependent, in chapter 6
the activity and the interaction of RSA was investigated in a different insect cell line, S2 cells
derived from embryos of Drosophila melanogaster, by doing some similar assays as
mentioned in chapter 5. In addition the effect of several kinase inhibitors on RSA activity
against S2 cells was investigated. Moreover, the potential binding partners for RSA on S2
cells were identified using RSA affinity chromatography.
The obtained results allowed us to draw a working hypothesis to explain the mode of action of
RSA in both cell lines (CF-203 and S2 cells).
22. 2Chapter 1
1.1. AGRICULTURE
Estimations by the United Nations have predicted that the global human population will be
over 7 billion people in 2012 and the population is expected to exceed 9 billion in 2050 and
10 billion in 2100. These increases in the human population are also increasing the
requirement for food. Agriculture is considered the main source of food and also the main
economy of the less developed countries. Agriculture is facing many problems which lead to
losses in the crop production, such as insects, weeds and diseases.
1.2. INSECTS
Insects are invertebrate animals belonging to the arthropods. They are one of the most diverse
organisms on the Earth. Insects include more than a million described species and represent
more than 90% of the different metazoan life forms on our planet. The ability of insects to
live in almost each environment makes them the most successful organisms occupying this
planet and in this way they can affect many aspects of our lives.
Insects are considered worldwide as one of the biggest problems in agriculture by attacking
and damaging different crops. Losses in agricultural production due to insect pests have been
estimated at 16% of the total production worldwide (Oerke et al., 1994). An attempt to
minimize crop losses due to insects was concerned by many researchers and entomologists.
Insect‟s bodies can be divided into three distinct parts: head, thorax and abdomen (Fig. 1.1).
The head carries the compound eyes and two antennae. While the thorax carries three pairs of
segmented legs and two or four wings. More than 60% of all known herbivorous insect
species are leaf-eating beetles (Coleoptera) or caterpillars (Lepidoptera) that cause damage
with their biting-chewing mouthparts (Fig. 1.2A) (Schoonhoven et al., 1998). In contrast,
Hemipteran insects have different piercing-sucking mouthparts that include a needle-like
stylet bundle consisting of two mandibular and two maxillary stylets (Cranston et al., 2003)
(Fig. 1.2B). Taxonomically, insects (Insecta class) belong to the subphylum Hexapoda, the
phylum Arthropoda within the Animal kingdom (Fig. 1.3). The class Insecta is subdivided
into orders, for example the order Lepidoptera and Hemiptera. Orders are divided into
families, families into genera, and genera are divided into species.
23. 3Chapter 1
Figure 1.1. Schematic representation of insect‟s morphology
Figure 1.2. (A) Schematic representation of Lepidopteran mouthparts (http://www.amentsoc.org/
insects/fact-files/mouthparts.html). (B) Schematic representation of Hemipteran mouthparts
(http://insected.arizona.edu/enforcers/resource/hemipteran.html)
24. 4Chapter 1
Figure 1.3. Taxonomy scheme of the insects used in this thesis. The taxonomy of the insects was
obtained from the following website (http://www.ncbi.nlm.nih.gov/Taxonomy/).
1.2.1. Hemiptera
Hemiptera (called also Rhynchota or true bugs) is one of the largest orders of insects. This
order consists of about 50.000-80.000 species. Many species of this order are considered
economically important pests, which are causing direct damage to plants by feeding or
causing an indirect effect by transmitting many plant virus diseases (Hogenhout et al., 2008).
Based on the differences in wing structure, the order of Hemiptera has been divided into two
distinct suborders. The first suborder is the Homoptera with insects where the front wing pair
may be uniformly membranous or stiffened throughout: good examples are aphids,
whiteflies, mealybugs, scale insects, froghoppers or spittlebugs, leafhoppers and treehoppers.
The second suborder is the Heteroptera (with the front wings clearly divided into two regions,
a hardened, leathery basal area and a membranous tip) such as shield bugs or stink-bugs,
capsid bugs, bedbugs, assassin bugs and water bugs.
Aphids are small hemimetabolous piercing-sucking insects, usually less than 5 mm, and
members of the Aphididae family, one family of the suborder Homopteran. Aphids are one of
the most destructive insect pests on the world agriculture (Pang et al., 2009). Aphids have a
pearlike shape and a pair of tubelike cornicles that can be found on the back of the abdomen.
This insect secretes honeydew which is known as a sugary liquid secreted through the anus.
25. 5Chapter 1
Wings are not always present; winged aphids are called "alates", while the wingless aphids
are known as "apterous” (Fig. 1.4).
Figure 1.4. Different forms of aphids: (A) wingless; (B) newborn nymph; (C) and (D) winged; (E)
nymph. (http://www.iranicaonline.org/uploads/files/Pests_Agricultural/pests_agric_fig_2.jpg)
1.2.1.1 Acyrthosiphon pisum
The pea aphid, A. pisum is a Hemipteran insect belonging to the Aphididae family. This aphid
is known to have a wide range of hosts from different legume species such as peas, alfalfa,
clover, and fresh beans, both snap and lima (Stoltz and McNea, 1982; Losey and Eubanks,
2000). Note that, the name “pea aphids” refers to the fact that pea crops are the major hosts in
the fields while other crops are considered as minor hosts (Hill, 1997). Pea aphids suck juice
from the phloem of their host plants by inserting their stylet (Fig. 1.2B) into the phloem
tissue. Then, the internal pressure inside the phloem helps to pump the sap into the aphid's
gut (Dixon, 1985). The wide host range and parthenogenic reproduction have made these
aphids one of the important migratory pests (Losey and Eubanks, 2000). The pea aphids have
a short and complex life cycle which includes two types of reproduction: the asexual and the
sexual reproduction (Fig. 1.5). Usually, eggs are laid in winter time and they enter a diapause
period. In the spring, these eggs hatch to asexual females which begin producing offspring
after 1-2 weeks following the hatching. After that, the aphids reproduce via parthenogenesis
by producing genetically identical nymphs that pass through four nymphal instars during
about 12 days before molting into an adult (Sharma et al., 1976; Blackman, 1987). In the fall,
the aphids develop to sexual females and males, and the mating results in overwintering eggs
26. 6Chapter 1
(Brisson and Stern, 2006). Pea aphids can easily be maintained in incubators to be used in
different laboratory bioassays.
Figure 1.5. The life cycle of the pea aphid (Brisson and Stern, 2006)
1.2.2. Lepidoptera
Lepidoptera is one of the largest orders of the class Insecta and belong to the most
widespread insects in the world. This order which is also called lepidopterans includes moths
and butterflies. The order of Lepidoptera consists of 47 superfamilies which consist of 128
families that have more than 180.000 species (http://www.ucl.ac.uk/taxome/). Insects of this
order are holometabolous and they are going through four stages in their life cycle: egg,
larva, pupa and adult (Powell, 2003) (Fig. 1.6). Among the Lepidoptera, adults commonly
feed on pollen or nectar while the larvae, called „caterpillars‟, are in many cases highly
phytophagous which makes this order one of the most destructive worldwide (Common,
1990).
27. 7Chapter 1
Figure 1.6. Different stages of Lepidoptera order. (A) egg; (B) larva; (C) pupa; (D) adult
http://ipm.ncsu.edu/ag271/peanuts/black_cutworm.html
1.2.2.1. Spodoptera littoralis
The cotton leafworm, Spodoptera littoralis (Fig. 1.7) belongs to the family Noctuidae and is
one of the most important lepidopterans in agriculture and horticulture, and has a wide host
range including at least 87 economically important plant species belonging to 40 families
distributed worldwide such as cotton, alfalfa, vegetables, maize, rice, soybeans, ornamentals,
weeds, etc. (Hill, 1987; Alford, 2003). This insect is one of the major insects in cotton fields
and can feed almost on all parts of cotton plants including the leaves, fruits, flower buds and
occasionally also on bolls. However, one of the problems to control this insect is its high
ability to develop relatively quickly resistance to most conventional insecticides. Each female
lays several hundred of eggs in clusters on the plant surface and covers them with orange-
brown hairs from the abdomen. The size of the egg is about 5 ± 2 mm diameter. Females of S.
littoralis have high fecundity and they can lay 2.000-3.000 eggs during 6-8 days. These eggs
hatch to larvae after 2-5 days after oviposition and immediately spread over the host plant.
The young caterpillars are gregarious but from 4th instar they become solitary and usually
they feed only at night and shelter in the soil during the day. Normally, the larvae develop
through six larval instars before inter the pupal stage. Pupation takes place in the soil inside a
loose cocoon and the pupae emergence and become adults (butterfly) after 7-10 days.
Butterflies are active at night and mate several times before laying eggs.
28. 8Chapter 1
The larvae of S. littoralis feed voraciously on almost all plant organs. Usually, they prefer
feeding on the young leaves, but when these leaves have been consumed the larvae can attack
also other parts such as stems, buds or pods. An infestation frequently leads to that all leaves
are devoured and plant development is affected by destroying growth points and flowers.
Figure 1.7. Larval stage of cotton leafworm, Spodoptera littoralis. Photo: M. Hamshou.
1.2.3. Insect gut
The insect gut is divided into three parts, the fore-, mid- and hindgut (Fig. 1.8A). The foregut
starts at the mouth and includes the cibarium, the pharynx, the esophagus, and the crop. The
latter is a storage organ in many insects and also serves as a site for digestion in others. In
most insects, foregut ends with the proventriculus, a valve to control the entry of food into the
midgut which is the main site for digestion and absorption of nutrients. The midgut consists
of the ventriculus, a simple tube from which blind sacs (gastric or midgut ceca) are branched.
The midgut epithelium of insects has several functions such as enzyme production, digestion,
and secretion (Chapman, 1998). These functions are probably because of the characteristic
structure of epithelial cells which form the midgut epithelium. Usually, the cytoplasm of the
epithelial cells has distinct regionalization in organelle arrangements, and as a consequence,
basal, perinuclear, and apical regions appear (Rost-Roszkowska et al., 2007; Rost-
Roszkowska and Undrul, 2008). Usually, the peritrophic membrane (PM), a film-like
anatomical structure is lining the midgut and separates the luminal contents into two places:
the endoperitrophic space and the ectoperitrophic space (Lehane, 1997). It is thought that the
PM plays a role to protect the gut surface from damage caused by abrasive food material and
to limit the access of microorganisms. In addition, it allows the transfer of liquid and digested
29. 9Chapter 1
substances to the midgut epithelial cells, but prevents the passage of larger food particles. The
columnar cells with a brush border (Fig. 1.8B) are the most common midgut epithelial cells
that are adjacent to the gut lumen. Although this membrane was found in most insects, it does
not occur in some insect orders such as Hemiptera, which are instead covered with
perimicrovillar membranes (PMVM) (Andries and Torpier 1982; Silva et al., 2004). The
domains of the microvilli are set in position by columns obliquely disposed between them
and the microvillar membrane (Fig. 1.8C) (Lane and Harrison, 1979). PMVMs maintain the
compartmentalization of digestion as an alternative to the peritrophic membrane (Ferreira et
al., 1988, Silva et al., 1995).
Figure 1.8. Schematic representation of insect gut compartments. (A) Different part of the midgut,
(B) Columnar cells, (C) Microvillus and (D) Glycocalyx: the carbohydrate moiety of intrinsic proteins
and glycolipids occurring in the luminal face of microvillar membranes (Terra and Ferreira, 2005).
30. 10Chapter 1
The glycoproteins and glycolipids on the luminal side of microvillar membranes are
decorated with a variety of carbohydrates (Fig. 1.8D) that play a role in mediating different
cellular and developmental events (Haltiwanger and Lowe, 2004). At the end of the midgut,
there is the sphincter or pylorus, a valve which locates between the midgut and the hindgut.
The hindgut consists of the ileum, colon and rectum and terminates with the anus (Fig. 1.8A).
The hindgut is involved in uptake of digested material, although to a lesser extent than the
midgut.
1.2.4. Insect cell lines
About half century ago, the first insect cell line was established from ovaries of the
diapausing silkmoth, Antheraea eucalypti (Grace, 1962). In the 50 years since that
achievement, many insect cell lines have been added to the list, to reach more than 500
established cell lines as depicted in Figure 1.9. This figure shows also that most of the insect
lines have been derived from Lepidoptera and Diptera (Lynn, 2001; Lynn, et al., 2005,
Smagghe, 2007).
Figure 1.9. The number of established invertebrate cell lines developed from 1962 to 2000
categorized by insect orders. (Source: Smagghe, 2007)
These cell lines were considered a useful research tool for screening of the biological efficacy
of novel pesticide candidates and their mode of action at the cellular level. In addition, cell
lines can provide large amounts of homogenous material in which the selected target sites are
directly present for the candidate insecticides. Insect cell lines have been derived from
different parts of the insect‟s body such as ovaries, embryos, hemocytes, imaginal discs, fat
body as well as from the midgut. These cells can easily be maintained in a laboratory by use
of specific culture medium. Recently, insect cell lines were used widely to investigate the
31. 11Chapter 1
toxicity of lectins and elucidate their mechanism of action. For example, the lepidopteran
midgut cell line (CF-203) was used to investigate the activity of different lectins (Smagghe et
al. 2005). Using CF-203 cells, Vandenborre et al. (2006) studied the interaction of a lectin
with receptor proteins in an attempt to determine the possible signal transduction pathways.
More recently, Shahidi-Noghabi et al. (2010a, 2011) did several assays using the same
midgut cells to determine the activity and the mode of action of Sambucus nigra agglutinin.
1.3. CROP PROTECTION
Up to date chemical insecticides are the most common compounds used to control insects.
These insecticides have been considered as one of the major factors involved in increasing
agricultural productivity in the 20th century. The world global pesticide market was about
US$ 40 billion in 2008 and it is expected to increase about 20 % in 2014 to reach 51 billion
(Fig. 1.10).
Figure 1.10. Global pesticide market by Segment (2008-2014)
http://www.bccresearch.com/report/biopesticides-market-chm029c.html
However, extensive use of chemical control has led to many problems including; (a) toxic
effects on humans, (b) developing resistance against these compounds by many pests, (c)
killing beneficial organisms such as pollinators, predators and parasitoids, (d) pesticide
residues in food, (e) harmful effects on nutrient cycling, (f) bad effects on soil, water and air
quality, and (f) reduction of biodiversity and impact on non-target species including some
mammals, birds, fishes, etc. through food chains. These problems pushed researchers to find
safer alternative methods to control pests.
32. 12Chapter 1
1.3.1. Current control strategies
In fact, the best way to control insects is the integrated pest management (IPM) which is
defined as using multiple tactics to control insect pests and to keep their abundance and
damage under the economic significance levels. IPM could include a combination of
practices such as the wisely use of pesticides, crop rotation, biological control and the use of
resistant plant varieties to suppress insect pest damage. The last category is one of the best
options which can be used and also includes the use of genetically engineered insect-resistant
crops.
The resistance of plants to insects is related to several defensive mechanisms which could be
separated to physical and chemical mechanisms. Proteins are one of most important
macromolecules which could be involved in the defensive mechanisms. Up to date, there are
many different proteins possessing an insecticidal activity which could be expressed in
transgenic plants including lectins, ribosome-inactivating proteins, protease inhibitors, α-
amylase inhibitors, arcelins, canatoxin-like proteins, ureases and chitinases (Carlini et al.,
2002; Vasconcelos et al, 2004; Karimi et al., 2010). The Bacillus thuringiensis (Bt) endotoxin
was the first protein that was expressed in tobacco plants (Vaeck et al., 1987). These plants,
engineered with truncated genes encoding Cry1A (a) and Cry1A (b) toxins, showed
resistance towards the larvae of the chewing tobacco hornworm Manduca sexta (Barton et al.,
1987, Vaeck et al., 1987). Since then, the transgenic crops that produce B. thuringiensis (Bt)
toxins are grown widely for pest control (Tabashnik et al., 2011). Two main problems were
faced using the Bt toxin based technology: it did not show protection towards sucking insects
and many insects developed a resistance to Bt toxin (Tabashnik et al., 1990; McGaughey and
Whalon, 1992; Ferre and Rie, 2002; Janmaat and Myers, 2003; Price and Gatehouse, 2008).
Because of these problems the interest grows to look for alternative strategies based on the
use of plant defence proteins such as lectins.
1.3.1.1. Lectins as bio-insecticidal agent
During the recent decades, many studies have focused on the investigation of the insecticidal
activity of different lectins, especially plant lectins, and the elucidation of their mechanism of
action while there were only very few studies on lectins from fungi (which is discussed in
fungal lectins part below). The insecticidal activity of plant lectins has been reported towards
different pest insects belonging to the orders Lepidoptera, Coleoptera, Diptera and
Homoptera (Vandenborre et al. 2009; Michiels et al. 2010). This activity of lectins and the
33. 13Chapter 1
potential of several plant lectins as insecticidal proteins was demonstrated both by in vitro
assays, using lectins incorporated into artificial diets (Sadeghi et al., 2009c; Shahidi-Noghabi
et al., 2010b), and in vivo assays, with transgenic plants expressing a foreign lectin gene
(Sadeghi et al., 2008; Shahidi-Noghabi et al., 2009).
1.3.1.1.1. Toxic effects of lectins towards Hemiptera
Plant lectins have been reported to possess insecticidal activity towards different insects
belonging to the Hemiptera order as demonstrated by using artificial diets incorporated with
lectins or transgenic plants expressing lectins. For instance, the lectin from Galanthus nivalis
(GNA) exerted toxic effects against different Hemipteran insects such as the pea aphid A.
pisum (Rahbé et al., 1995), the glasshouse potato aphid Aulacorthum solani (Down et al.,
1996), and the red cotton bug Dysdercus cingulatus (Roy et al., 2002) when it was
incorporated into the artificial diet. In addition, transgenic plants expressing GNA also
affected the growth and survival of some insects belonging to Hemiptera. For example,
genetically modified rice plants showed insecticidal activity against the green rice leafhopper
Nephotettix virescens (Ramesh et al., 2004), the brown planthopper Nilaparvata lugens (Saha
et al., 2006b) and the small brown planthopper Laodelphax striatellus (Sun et al., 2002).
Moreover, the peach aphid Myzus persicae was shown to be sensitive to the Allium sativum
lectin when the lectin was added to the artificial diet (Sauvion et al., 1996) or expressed in
tobacco plants (Dutta et al., 2005b).
1.3.1.1.2. Toxic effects of plant lectins towards Lepidoptera
Many plant lectins have been reported to affect insect growth, development, and fecundity of
a wide range of Lepidopteran insects when these insects were fed on an artificial diet
supplemented with lectins and/or on transgenic plants overexpressing the lectin genes.
For example, the cotton bollworm Helicoverpa armigera was found to be affected by
different plant lectins isolated from Galanthus nivalis, Triticum aestivum, Canavalia
ensiformis, Arachis hypogea, Artocarpus integrifolia, Cicer arietinum and Lens culinaris
when the larvae of H. armigera were fed on an artificial diet containing different
concentrations of these lectins (Shukla et al., 2005). Furthermore, larvae of the European corn
borer (Ostrinia nubilalis) were found to be sensitive to lectins from Triticum aestivum,
Ricinus communis and Bauhinia purpurea (Czapla & Lang, 1990).
In addition, transgenic plants expressing lectins (mainly GNA) exerted an insecticidal activity
towards different insects from the order Lepidoptera. For instance, GNA expressed in tomato,
34. 14Chapter 1
tobacco, rice and sugarcane showed toxic effects towards Lacanobia oleracea (Wakefield et
al., 2006), Helicoverpa assulta (Zhang et al., 2007), Chilo suppressalis (Loc et al., 2002) and
Eoreuma loftini (Setamou et al., 2002). Furthermore, transgenic rice expressing Allium
sativum leaf agglutinin exhibited entomotoxic activity against different sap-sucking pests
(Yarasi et al., 2008). In addition transgenic tobacco plants expressing A. sativum lectin or
leek lectin demonstrated entomotoxic activity against S. littoralis (Sadeghi et al., 2007;
Sadeghi et al., 2009a).
1.3.1.1.3. Interaction of lectins with receptors in insect
The biological activity of lectins depends on their ability to bind carbohydrates which are all
present on the surface of cells, such as the epithelial cells of animal digestive tracts (Villalobo
and Gabius, 1998). The importance of lectin binding to a sugar moiety of a glycosylated
protein in the insect gut has been suggested to be the prerequisite factor for the insecticidal
activity of any lectin (Peumans and Van Damme, 1995a; Peumans and Van Damme, 1995b).
For example, toxicity of Phaseolus vulgaris agglutinin (PHA) on the midgut epithelial cells
of the bruchid Callosobruchus maculatus was proposed to depend on the binding of PHA to
these cells (Gatehouse et al., 1984). Moreover, the correlation between binding and
insecticidal activity of PHA against different insects was reported (Habibi et al., 1998; Habibi
et al., 2000; Fitches et al., 2001; Bandyopadhyay et al., 2001). In contrast, lack of binding of
PHA to the midgut cells of bean weevil (Acanthoscelides obtectus) could explain the non-
toxic effect of PHA towards this insect (Gatehouse et al., 1989). In fact, the correlation
between the binding of the lectins and their insecticidal activity is not general for all lectins.
For instance, detailed studies on the mechanisms of two lectins from Sambucus nigra (SNA-I
and SNA-II) on the insect midgut CF-203 cells, revealed that both lectins did not bind the
cells but they got internalized in the cells which resulted in strong toxicity (Shahidi-Noghabi
et al., 2011). The importance of the carbohydrate-binding domain for the insecticidal activity
has been demonstrated by two different methods (i) mutation of Griffonia simplicifolia lectin
to eliminate the carbohydrate-binding activity reduced the toxicity of this lectin towards the
cowpea bruchid, C. maculatus (Zhu-Salzman et al., 1998); (ii) incubation of different lectins
with their specific sugar reduced the binding and toxicity of these lectins on different cell
lines (Kuramoto et al., 2005).
35. 15Chapter 1
1.4. INSECT GLYCOSYLATION PATTERNS
Membrane proteins were reported to serve as transport systems, light-transducing agents,
antigens and receptors. Plasma membranes contain carbohydrates as glycoproteins and
glycolipids. In general, glycosylation occurs on the extracellular surface of the plasma
membrane. Glycosylation is defined as a covalent attachment of an oligosaccharide chain to a
protein and is considered to be a very common protein modification. The composition of the
carbohydrate chain is very diverse and can modify the characteristics of a protein. The two
major forms of this protein modification are N-glycans and O-glycans which refers to the
type of glycosidic linkage of this carbohydrate structure to the amino acids Asn and Ser/Thr,
respectively. Glycosylation of proteins can mediate different processes such as subcellular
localization, protein quality control, cell-cell recognition and cell-matrix binding events in
addition to other rules which are not fully understood. In fact, most studies on glycobiology
have focused on mammals although insect glycobiology is a promising research field because
they are the most diverse organisms and have a wide genetic diversity. Up to date, almost all
information concerning glycobiology in insects was obtained from studies with the fruit fly,
Drosophila melanogaster (Diptera).
Studies on D. melanogaster have shown that glycans could affect developmental processes as
demonstrated by using lectins to study the variation of glycosylation as a function of organ,
cell type, and developmental stage in this insect (Fredieu and Mahowald, 1994; D'Amico and
Jacobs, 1995). Moreover, glycosylation was reported to contribute to the function(s) of some
proteins with important roles in development (O'Tousa, 1992; Kaushal et al., 1994)
Drosophila proteins were shown to be decorated with high-mannose oligosaccharides and
core fucosylated pauci-mannose glycans as demonstrated by different N-linked glycans
studies (Seppo and Tiemeyer, 2000; Fabini et al., 2001; Sarkar et al., 2006). Furthermore, the
N-glycan profile of the fly was found to change according to the developmental stages which
suggests specific roles of certain glycan structures during different stages of development
(Seppo and Tiemeyer, 2000; Aoki et al., 2007; Ten Hagen et al., 2009). Recently a protein
modified by a mucin type O-linked glycosylation was identified from Drosophila
(Schwientek et al., 2007; Tian and Hagen, 2009). The recent progress in using lectins in
glycoproteomics and insect glycobiology will provide new insights in the interactions
between lectins and insects, which in turn will help to better understand the mode of action
behind the lectin activity.
36. 16Chapter 1
1.4.1. Gal/GalNAc Linkage residues
Compared with the wide heterogeneity observed in most animals, insects seem to synthesize
a surprisingly low number of very simple O-glycans. So far, studies conducted on several
Lepidopteran cell lines suggested that the O-glycosylation in insects was restricted to
GalNAc-α-Ser/Thr and Galβ1–3GalNAc-α-Ser/Thr (Thomsen et al., 1990; Kramerov et al.,
1996; Lopez et al., 1999; Maes et al., 2005; Garenaux et al., 2011). In addition, the most
abundant O-glycan structure in Drosophila is the mucin type O-glycosylation. As shown in
figure 1.11, this type of glycosylation involves in the addition of GalNAc to Ser/Thr to form
the Tn antigen (GalNAcα1-S/T), often extended with galactose (Gal) (Tian and Hagen,
2009).
Figure 1.11. Biosynthesis of the most common mucin-type O-glycans in D. melanogaster (Tian and
Hagen, 2009).
37. 17Chapter 1
Moreover, investigation of the involvement of glycosyltransferases in complex-type N-
glycosylation in different Lepidopteran insect cell lines suggested the ability of these cell
lines to synthesize complex type carbohydrate chains containing GalNAc β14GlcNAc units
(Van Die et al., 1996; Tran et al., 2012). The presence of fucosylated, sialylated, hybrid,
biantennary complex, and triantennary complex glycans in Drosophila embryos was
demonstrated (Varki et al., 2008). Interestingly, some lectins which can recognize and bind to
Gal/GalNAc have been reported to possess high insecticidal activity, such as lectin from
Sambucus nigra (Shahidi-Noghabi et al., 2010b) and Glechoma hederacea lectin (Wang et
al., 2003).
1.5. APOPTOSIS
For all the living organisms, including the life cells in the earth and universe itself, there is a
time to live and afterwards a time to die. There are two ways in which cells die as a response
to a variety of stimuli, such as toxins, genotoxic compounds, tumor necrosis factor and
various environmental stresses: (i) Killing the cells by injury or disease, which is
uncontrolled cell death or (ii) Programmed Cell Death (PCD) or apoptosis, which is a
regulated cell suicide. Eventually, the term apoptosis was used in order to describe the
morphological processes that lead to controlled cellular self-destruction. This term was first
used in a publication by Kerr et al. (1972).
Apoptosis is a normal component of the development and health in the multicellular
organisms by which cells undergo death to control cell proliferation or in response to DNA
damage. A good example for the involvement of apoptosis in animal development is a
massive cell death in the interdigital mesenchymal tissue to form free and independent digits
(Zuzarte-Luis and Hurle, 2002). Another example, during nervous system development,
about 1.5 times the adult number of neurons will die by apoptosis in later stages when the
adult nervous system is formed (Hutchins, 1998). The apoptosis has several characteristics
such as shrinkage of cells, chromatin condensation, blebbing, formation of membrane-bound
apoptotic bodies that contain organelles, cytosol and nuclear fragments (Fig. 1.12). And
finally the cells suicide and died (Gewies, 2003; Ma et al., 2005). Three different mechanisms
of apoptosis have been described. A first mechanism occurs as a response to internal by
signals in a cell such as Bcl-2, Apaf-1 (apoptotic protease activating factor-1), Bax,
cytochrome c, caspase 9, ATP, etc. A second mechanism is caused by external signals such
38. 18Chapter 1
as Fas, FasL, TNF, TNF receptor, etc. and a third mechanism is triggered by toxic factors
(Ma et al., 2005).
Apoptosis is of widespread biological significance and could be involved in several
biological processes such as development, differentiation, proliferation, regulation and
function of the immune system and in the removal of defect and therefore harmful cells
(Gewies, 2003). Thus, dysfunction or dysregulation of apoptosis can result in a variety of
pathological conditions. For instance, defects in the apoptotic process can cause cancer,
autoimmune diseases and spreading of viral infections, while excessive apoptosis can
enhance neurodegenerative disorders, AIDS and ischaemic diseases (Fadeel, 1999).
Moreover, apoptosis is also considered as a defense mechanism against virus infection
directly interfering with virus multiplication (Clem and Miller, 1993) and also against
bacterial pathogens by eliminating the infected cells via programmed cell death (Böhme and
Rudel, 2009).
Actually, the central executioners of the apoptotic signaling pathway are caspases which are
activated in most cases of apoptotic cell death (Bratton, 2000; Olsson and Zhivotovsky,
2011).
Figure 1.12. Cellular changes during apoptotic cell death. The changes include cellular shrinking,
chromatin condensation and margination at the nuclear periphery with the eventual formation of
membrane-bound apoptotic bodies that contain organelles, cytosol and nuclear fragments and are
phagocytosed without triggering inflammatory processes. The photo is modified from Gewies (2003).
It is worth mentioning that there are caspase-independent apoptosis pathways which could
depend on calpains, cathepsins, endonucleases, and other proteases. These proteins can
initiate and execute programmed cell death that can be regulated by several cellular
39. 19Chapter 1
organelles such as mitochondria, lysosomes, and the endoplasmic reticulum (ER), which can
work together or independently (reviewed by Bröker et al., 2005).
About 50 years ago, the involvement of apoptosis in insect development has been reported by
Lockshin and Williams (1964). The first ecdysone peak during metamorphosis of the wild
silkmoths and the tobacco hawkmoth induces apoptotic degeneration of the larval
intersegmental muscles, proleg motoneurons, and labial glands (Lockshin and Williams,
1964; Lockshin and Zakeri, 1994). Moreover, apoptosis can be induced as result of the
decrease in the ecdysone titer shortly before hatching degeneration of abdominal neurons and
intersegmental muscles (Truman, 1984). Important changes in food habits between larval and
adult stages show large modifications in the digestive tract. For instance, the larval midgut of
the greater wax moth, Galleria mellonella, undergoes apoptosis during metamorphosis (Uwo
et al., 2002). Moreover, apoptosis of the larval midgut of Heliothis virescens was correlated
with higher caspase expression shortly before and after pupation (Parthasarathy and Palli,
2007). A recent study demonstrated that apoptosis is a fundamental host defense mechanism
against Parachlamydiaceae in insect cells (Sixt et al., 2012).
It is worth mentioning that Apoptosis which is reported in the salivary gland of Apis mellifera
larvae was found to lie between the classical apoptosis and autophagy because it exhibited
some characteristics of both phenomena (Silva-Zacarin et al., 2007).
Two types of PCD have been reported during Drosophila development: (i) apoptosis which is
characterized by membrane blebbing, nuclear condensation and DNA fragmentation, and (ii)
autophagy which is distinguished by the destruction of the whole tissues and the presence of
autophagic vacuoles (Abrams et al., 1993). Interestingly, PCR cloning studies as well as the
analysis of the complete Drosophila euchromatic genomic sequence showed that there are
insect homologs for many of the mammalian PCD genes (Rubin et al., 2000; Vernooy et al.,
2000). Drosophila was considered a good and easy way to investigate the function of these
PCD genes in vivo (Hay and Guo, 2006).
The release of cytochrome c from the mitochondria by various apoptotic stimuli initiates the
major caspase activation pathway(s) in mammalian cells (He et al., 2000; Arnoult et al.,
2002; Jiang and Wang, 2004). In insects (Fig 1.13), cytochrome c was found to be involved
in apoptosis of many lepidopteran cell lines such as Sf9 cells (Sahdev et al. 2003), Sl-1 cells
(Malagoli et al., 2005) and LdFB cells (Shan et al., 2009). In contrast, the majority of studies
40. 20Chapter 1
on Drosophila showed that there is no evidence for the involvement of cytochrome c in
apoptosis of this insect (Liu et al., 2012).
Figure 1.13 Model for the role of cytochrome c during insect cell apoptosis (from: Liu et al., 2012).
1.5.1. The insect caspases
Caspases (cysteine aspartate-specific proteinases) are one of the main executors of the
apoptotic process in mammals and insects. They belong to a family of cysteine proteases and
exist within the cell as inactive pro-forms or zymogens. These zymogens can be cleaved to
form active enzymes following the induction of apoptosis. There are two types of apoptotic
caspases, based on their place of entry into the cell death pathway: initiator (apical) caspases
and effector (executioner) caspases. The prodomain of the initiator caspases contains the
death effector domain (DED) in procaspase-8 and -10, or the caspase recruitment domain
(CARD) in procaspase-2 and procaspase-9 (Thornberry and Lazebnik, 1998; Earnshaw et al.,
1999; Fuentes-Prior and Salvesen, 2004). Both DED and CARD are involved in procaspase
activation and downstream caspase-cascade regulation through protein-protein interactions
(Fuentes-Prior and Salvesen, 2004; Ho and Hawkins, 2005). The activation of caspases is
usually occurring through two pathways: the death signal-induced or death receptor-mediated
pathway and the stress-induced or mitochondrion-mediated pathway (i.e. a caspase-9-
dependent pathway) (Fan et al., 2005).
41. 21Chapter 1
In mammals, the death receptors, such as Fas or TNF, can specifically recognize cell death
signals, such as FasL (Fas ligand) or TNF (tumor necrosis factor). This binding activates the
death receptors. Then, Fas can bind to the Fas-associated death domain (FADD) (or TNFR-
associated death domain, TRADD) and cause FADD aggregation and the emergence of
DEDs which interact with the DEDs in the prodomain of procaspase-8/-10. The result of this
interaction is formation of the death-inducing signal complex (DISC) that activate
the initiator caspases-8, -9, -10 (Fig. 1.14) (Boatright and Salvesen, 2003; Alenzi et al.,
2010). Subsequently, the initiators activate the effector caspases, caspase-3, -6, -7 (Boatright
and Salvesen, 2003). Then, the effector caspases cleave key cellular substrates such as protein
kinases, signal transduction proteins and DNA repair proteins (Fischer et al., 2003).
Apoptosis can also occur via intrinsic pathways which are triggered in response to a wide
range of intracellular signals, such as oncogene activation and DNA damage. Those
intracellular signals are altering the permeability of the mitochondrial outer membrane which
in turn leads to the release of several proteins to the cytosol, such as Smac/Diablo and
cytochrome c. Cytochrome c forms an apoptosome, a catalytic multiprotein platform that
activates caspase-9. Subsequently, activation of caspase-8 and/or caspase-9 leads to activate
the effector caspase-3, -6 and -7 (Fig. 1.14) (Czerski and Nuñez, 2004).
Figure 1.14. Schematic representation of caspase-dependent apoptosis pathways in mammals and the
main regulating factors in apoptotic pathways (Fan et al., 2005).
42. 22Chapter 1
Various molecules were reported to regulate the activation and inactivation of caspases such
as IAP, Bcl-2 family proteins, calpain, Ca2+
, Gran B and cytokine response modifier A (Crm
A) (Fig. 1.14) (Launay et al., 2005).
Caspases have been characterized and studied well in mammals but they are less documented
in insects. In fact, the insect caspases were described mainly in D. melanogaster (Kumar and
Doumanis, 2000; Cooper and Granville, 2009) and recently in Lepidopteran insects
(Courtiade et al., 2011). In Drosophila, some caspases were reported to have a homologue
with mammalian caspases, while others have none. For instance, Dredd (a Drosophila
caspase) has similarity with mammalian caspase-8 and Dronc (a Drosophila caspase) is a
homologue of the mammalian caspase-9 and the human caspase-2 (Kumar and Doumanis,
2000). Strica (a Drosophila caspase) has no similarity to any other characterized motifs such
as CARD and death inducing domain, DID.
Interestingly, some homologues of Drosophila proteins involved in apoptosis have been
recognized in other insects. Aedes aegypti Dredd (AeDredd) was found to have the highest
sequence similarity with Drosophila Dredd and with human caspase-8 (Cooper et al., 2007a).
Aedes Dronc (AeDronc), is a homologue of the Drosophila Dronc (Cooper et al., 2007b).
Homologues of Drosophila Strica/Dream have been identified in the genome of both A.
aegypti and A. gambiae (Bryant et al., 2008). In addition, the homologeus of Drosophila
Dredd have been identified in Tribolium castaneum (Zou et al., 2007).
In Lepidoptera, several caspases have been identified. Sf-caspase-1 was the first insect
caspase identified from the lepidopteran Spodoptera frugiperda. This caspases was found to
be similar to Drosophila Drice and mammalian caspase-3 (Ahmad et al., 1997). Later, a
caspase called Sl-caspase-1 was found in S. littoralis cells which showed similarity with Sf-
caspase-1 (Liu et al., 2005). Moreover, Tn-caspase-1 was characterized in Trichoplusia ni
and found to be the main effector caspase in T. ni cells (Hebert et al., 2009). Recently, Hearm
caspase-1, an effector caspase identified from the cotton bollworm, Helicoverpa armigera,
has been found to be homologous to Sf-caspase-1 and Drosophila Drice (Yang et al., 2008)
and the homologue of Drosophila Dredd was identified in Bombyx mori (Xia et al., 2008). In
a recent study, 63 caspase genes were identified from 27 different lepidopteran species.
Phylogenetic analyses demonstrated that Lepidoptera possess at least 5 caspases (Courtiade et
al., 2011). Lep-Caspase-1, -2 and -3 were found to be putative effector caspases, while Lep-
Caspase-5 and -6 are reported to be putative initiator caspases in homology to Drosophila
43. 23Chapter 1
caspases. However, these caspases need further study to clarify the exact function and their
potential interactions (Courtiade et al., 2011). Figure 1.15 shows a comparative analysis of
the different homologues of caspases in the apoptotic pathway in mammals, Drosophila and
Lepidoptera.
Figure 1.15. Apoptotic pathway in mammals, Drosophila and Lepidoptera. Homologs of caspases
and caspase regulators across species are indicated by the same color. Initiator and effector caspases
are colored in blue and red respectively. The death receptor is colored in grey, the adaptor protein in
orange, the protein forming the apoptosome in yellow, the apoptotic inducers in purple, and the
caspase inhibitors in brown. (from: Courtiade et al., 2011).
44. 24Chapter 1
1.6. FUNGAL LECTINS: their toxicity and antiproliferative activity
Lectins are carbohydrate-binding proteins of non-immune origin possessing at least one non-
catalytic domain, which binds reversibly and non-covalently to mono- or oligosaccharides,
glycoproteins and glycolipids (Goldstein et al., 1980; Peumans and Van Damme, 1995a).
More than a century ago the first lectin was described by Stillmark who discovered lectin
activity in the seeds of castor tree, Ricinus communis (Stillmark, 1888). Since then, many
new lectins from various sources have continuously been added to the list of carbohydrate-
binding proteins. Due to their ability to bind carbohydrates, most of these proteins can also
agglutinate erythrocytes, a reaction which can be inhibited by using a specific sugar (Sumner
and Howell, 1936). Lectins are ubiquitously distributed in nature and can be found in plants,
fungi, bacteria, viruses, invertebrates and vertebrates (Vandenborre et al., 2009; Khan and
Khan, 2011; Vasta and Ahmed, 2008; Hartmann and Lindhorst, 2011). They are valuable
proteins not only because they are found in all organisms, but especially because their
reversible interaction with specific carbohydrates allows them to bind to glycoconjugates that
play an important role in cell physiology. All these properties have made lectins as one of the
most studied groups of proteins which are used as tools in biological and biomedical
research, especially in studies related to cell-cell interactions, cancer invasion and metastasis,
inflammation, and immunology.
In the past decades plant lectins have been studied in much more detail than any of the lectins
from other sources. Many plant lectins have been found in storage tissues where they
represent 0.1–10% of the total protein in the tissue. Therefore it has been proposed that these
lectins could serve as plant storage proteins (Van Damme et al., 1998). Furthermore, owing to
their ability to recognize specific carbohydrates it was suggested that these lectins may act as
defense proteins (Peumans and Van Damme 1995a). This hypothesis was shown to be correct
for several plant lectins (Michiels et al., 2010; Vandenborre et al., 2011b).
Lectins from fungi are far less documented than the plant lectins. Phallin was the first fungal
lectin that was discovered in Amanita phalloides in 1891 (Kobert, 1893) and later in 1910,
the second fungal lectin was reported from the mushroom Amanita muscaria (Ford, 1910).
To date more than 350 fungal lectins have been reported. The majority of these lectins was
detected in mushrooms (which can be defined as a macrofungi with a distinctive fruiting
body) and the rest was isolated from microfungi (which can be distinguished
from macrofungi only by the absence of a large fruiting body). Lectins have been isolated
45. 25Chapter 1
from the orders Agaricales, Boletales, Russulales, Cantharellales, Atheliales, Polyporales and
Thelephorales.All these orders belong to the class Agaricomycetes and the phylum
Basidiomycota. In addition, a few fungal lectins were purified from the orders Eurotiales,
Helotiales, Pezizales, Sordariales and Xylariales which belong to different fungal classes
within the phylum Ascomycota. Both phyla Basidiomycota and Ascomycota belong to the
subkingdom Dikarya within the kingdom of Fungi (Fig. 1.16).
Figure 1.16. Overview of the taxonomy of fungi from which lectins were isolated and will be
discussed in this review. The taxonomy of the fungi was obtained from the following website
(http://www.ncbi.nlm.nih.gov/Taxonomy/).
Fungal lectins have been reviewed in several recent papers (Guillot and Konska, 1997; Wang
et al., 1998; Konska, 2006; Singh et al., 2010; Khan and Khan, 2011a, Singh et al., 2011).
This chapter will give an overview only of those fungal lectins that were shown to possess
toxic properties and/or antiproliferative activity (Table 1.1).
50. 30Chapter 1
1.6.1. Basidiomycota
1.6.1.1. Lectins from the fungal order Agaricales
1.6.1.1.1. Agaricus arvensis lectin
A. arvensis lectin (AAL) is an inulin specific lectin purified from the dried fruiting bodies of
the wild edible mushroom A. arvensis. AAL has a molecular weight of 30.4 kDa and is
composed of two subunits of 15.2 kDa each (Zhao et al., 2011). The lectin exhibits potent
antiproliferative activity towards HepG2 and MCF-7 tumor cells with an IC50 of 1.64 and 0.82
μM, respectively.
1.6.1.1.2. Agaricus bisporus lectin
Four A. bisporus lectins (ABL) were found in the common commercial golden white
mushroom A. bisporus. They have molecular weights ranging between 64 and 85 kDa, and
are made up of identical subunits of 16 kDa (Presant and Kornfeld, 1972; Ahmad et al., 1984;
Sueyoshi et al., 1985). The biological activity of all these ABLs cannot be inhibited by any
simple sugar but is inhibited by Gal β-1,3-GalNAc (Yu et al., 1993).
Incubation of ABL with different cell lines (HT29 human colorectal carcinoma cells, Caco-2
human colorectal cancer cells, human breast cancer MCF-7 cells, and rat mammary
fibroblasts Rama-27 cells) revealed an inhibitory effect of the lectin in all these cells in a
dose-dependent manner. For instance, 50% inhibition of HT29, MCF-7 and Rama-27 cells
was achieved by 3, 5 and 25 μg/ml ABL, respectively, while this value was more than 50
μg/ml for Caco-2 cells (Yu et al., 1993). ABL also exerted a dose-dependent proliferation
inhibitory effect on human ocular fibroblasts. This inhibition was recorded to be 40% when
ABL was dosed at 100 μg/ml (Batterbury et al., 2002). It was shown that FITC-ABL was
bound to the cell surface and was then internalized in the cells and accumulated around
the nuclear envelope. Furthermore, ABL induced a strong antiproliferative activity against
human retinal pigment epithelial cells with an inhibition of 80% at 60 µg/ml ABL. It was
proposed that ABL could block the antigenic sites which resulted in the inhibition of cell
proliferation (Kent et al., 2003). ABL also caused lymphocyte (T cells) death in a dose- and
time-dependent manner with a reduction in the cell viability of about 50% after a 2h
incubation with 100 nM lectin. Most of the cells died after 24 h (Ho et al., 2004).
51. 31Chapter 1
1.6.1.1.3. Agrocybe aegerita lectin
A. aegerita lectin was isolated from the fruiting bodies of the mushroom A. aegerita. The
lectin is a homodimeric protein and consists of two subunits of 15 kDa. The activity of A.
aegerita lectin was inhibited by lactose and some glycoproteins such as bovine submaxillary
mucin, glycophorin A, and hog gastric mucin (Sun et al., 2003). The lectin agglutinates
erythrocytes of all human types (A, B and O) and 12 different animal species.
The A. aegerita lectin was reported to have high inhibitory activity towards human and mouse
tumour cells. For instance Zhao et al. (2003) reported a strong inhibitory effect of the A.
aegerita lectin against seven different tumour cell lines (SW480, HeLa, SGC-7901, MGC80-
3, BGC-823, HL-60 and S-180 cells). The effects of A. aegerita lectin in all these cell lines
were dose-dependent with inhibition effects between 42.8% and 82.6% as determined by
MTT assay when the lectin was dosed at 100 µg/ml (Zhao et al., 2003). Moreover, in vivo
studies showed that when A. aegerita lectin was injected into tumour-bearing mice the lectin
reduced the tumour growth by 36.36%, which also significantly reduced the death ratio of the
treated group by 80% compared with the control group (Zhao et al., 2003). Interestingly, the
A. aegerita lectin exerted toxicity towards mice with an LD50 value of 15.85 mg/kg (Sun et al.,
2003).
It was shown that the activity of the A. aegerita lectin in HeLa cells was due to apoptosis
induction which depends mainly on the internalization of the lectin into the cells and its
nuclear localization (Liang et al., 2009). In addition, DNase activity was also proposed as a
mechanism behind the A. aegerita lectin activity (Zhao et al., 2003). Similar to the native A.
aegerita lectin, the recombinant A. aegerita lectin also induced apoptosis in HeLa cells (Yang
et al., 2005a).
In addition to the activity of the A. aegerita lectin on tumour cells, the lectin showed antiviral
activity towards tobacco mosaic virus (TMV) (Sun et al., 2003). The 50% inhibition dose of
the lectin for TMV infection was determined to be 35 ± 5 µg/ml. To explain the mode of
action of the A. aegerita lectin on TMV it was suggested that the lectin attaches to TMV
which leads to blocking of the infection sites (Sun et al., 2003).
1.6.1.1.4. Agrocybe cylindracea lectin
A lectin named ACL was purified from the fruiting bodies of the edible mushroom A.
cylindracea (Yagi et al., 1997). ACL was found to be a heterodimeric lectin with a molecular
52. 32Chapter 1
weight of 31.5 kDa and has specificity towards lactose, sialic acid and inulin. ACL was
reported to have potent mitogenic activity towards mouse splenocytes (Wang et al., 2002a).
ACL exhibited potent anti-nematode toxicity against two plant parasitic nematodes
Ditylenchus dipsaci and Heterodera glycines (Zhao et al., 2009a). The effect of ACL was
concentration-dependent as well as time-dependent with an LC50 of 1.4 mg/ml when D.
dipsaci was incubated for 48 h with the lectin. A 4.5-fold lower toxicity of ACL was recorded
on H. glycines (LC50 = 6.3 mg/ml) (Zhao et al., 2009a). The toxic effect of ACL was reduced
about 40 % in both nematodes after adding a specific sugar (lactose).
1.6.1.1.5. Amanita virosa lectin
A 36 kDa lectin was isolated from the fruiting bodies of the mushroom A. virosa. The lectin
was characterized as a homodimeric protein composed of two subunits with a molecular mass
of 18 kDa. The activity of this lectin was not inhibited by any simple sugar (Antonyuk et al.,
2010). This lectin exerted a cytotoxic effect towards CEM T4 and Jurkat human cells with
respective LD50 values of 0.72 and 0.44 μg/ml, respectively, while less toxicity was found in
the mammalian leukemia L1210 cells, the LD50 being 3.42 μg/ml (Antonyuk et al., 2010).
1.6.1.1.6. Armillaria luteo-virens lectin
A lectin called ALL has been found in dried fruiting bodies of the A. luteo-virens mushroom.
It is a dimeric protein with a molecular weight of 29.4 kDa. ALL shows specificity towards
inulin (Feng et al., 2006). The lectin showed antiproliferative activity against MBL2, L1210
and HeLa tumor cells with IC50 values of 2.5, 5, and 10 μM, respectively.
1. 6.1.1.7. Clitocybe nebularis lectin
Different lectins have been found in the C. nebularis fruiting bodies. The molecular mass of
these lectins ranged between 15.5 and 31 kDa. These proteins show specificity mainly for
asialofetuin and lactose (Pohleven et al., 2011). CNLs belong to the ricin B-like lectin
superfamily (Pohleven et al., 2009).
CNL was reported to have antiproliferative activity towards leukemic Mo-T cells as
determined by the MTS assay (Pohleven et al., 2009). The effect of CNL was dose-dependent
and the reduction in cellular proliferation was about 60 % at 100 µg/ml CNL. Interestingly the
activity of CNL was abolished after preincubation of the lectin with its specific sugar (lactose)
which most probably means that binding of CNL to a specific sugar is the first step in starting
53. 33Chapter 1
the biological effect of the lectin (Pohleven et al., 2009). A similar inhibition was reported in
Jurkat cells after incubation with recombinant CNL (Pohleven et al., 2012).
Feeding of the nematode Caenorhabditis elegans on Escherichia coli expressing CNL
inhibited the development of the larvae by approximately 50% and none of these larvae
developed to adult whereas about 80% of these larvae became an adult in the control
treatment (Pohleven et al., 2012).
CNL exhibits insecticidal activity towards different insects. Feeding of the fruit fly
(Drosophila melanogaster) on a diet containing CNL resulted in a significant mortality with
an LC50 about 48 μg/ml. In addition, CNL showed an important anti-nutritional effect towards
the Colorado potato beetle (Leptinotarsa decemlineata) and this effect was concentration-
dependent. For example feeding the larvae on 0.02% CNL for 10 days reduced the larval
weight about 50% compared to the control larvae (Pohleven et al., 2011). Moreover a 10-fold
higher toxicity towards D. melanogaster was observed with another lectin isolated from
Clitocybe nebularis (called CnSucL) but this lectin did not show any toxic effect in L.
decemlineata (Pohleven et al., 2011).
Feeding of Aedes aegypti on a diet containing E. coli BL21 (DE3) cells expressing CNL
reduced the survival of second instar larvae for about 80% (Bleuler-Martínez et al., 2011).
The same authors also showed that CNL has toxicity towards the amoeba Acanthamoeba
castellanii.
1.6.1.1.8. Coprinopsis cinerea galectin (CGL2)
Several lectins are present in the fruiting bodies of the mushroom C. cinerea. They are called
CGL1, CGL2 and CGL3 and are genetically related to family of β-galactoside-binding lectins
(Cooper et al., 1997; Boulianne et al., 2000; Walti et al., 2008).
Both CGL1 and CGL2 show nematotoxic activity towards C. elegans (Butschi et al., 2010).
Practically, L1 larvae of C. elegans were fed on a diet containing E. coli cells expressing
either the CGL1 or the CGL2 proteins. After 72h the number of L4 larvae was recorded. Only
10 ± 10% larvae in both treatments reached the L4 stage while all the larvae in the control
treatment became L4. Further analysis on CGL2 showed that the effect was dose-dependent
with an LD50 value of 350 mg/ml (Butschi et al., 2010).
The toxicity of CGL2 was dependent on its ability to bind carbohydrate moieties mainly on
the intestinal epithelium of C. elegans while no activity was detected with the mutant CGL2
54. 34Chapter 1
protein (W72G) which does no longer possess β-galactoside binding activity (Butschi et al.,
2010).
In addition to the anti-nematode activity, CGL2 also has anti-insect activity. Feeding of A.
aegypti on recombinant CGL2 expressed in E. coli reduced the larval survival for about 80%
(Künzler et al., 2010).
1.6.1.1.9. Flammulina velutipes lectin
FVL is a hemagglutinin composed of one subunit of 12 kDa found in the fruiting bodies of
the mushroom F. velutipes. The hemagglutinating activity of FVL was inhibited by
lactoferrin, a milk glycoprotein (Ng et al., 2006). FVL exerted a dramatic antiproliferative
activity against L1210 cells with an IC50 of 13 μM. Moreover 40 μM FVL inhibited the
cellular proliferation completely.
1.6.1.1.10. Grifola frondosa lectin
The lectin named GFL was isolated from the fruiting bodies of the mushroom G. frondosa.
GFL has high affinity for GalNAc and a molecular mass between 30-52 kDa (Kawagishi et
al., 1990). More recently, Nagata et al. (2005) extracted another lectin from G. frondosa with
a molecular weight of 24 kDa. In contrast to the GFL isolated by Kawagishi et al. (1990), the
activity of GFL was not affected by any monosaccharide but was only inhibited by porcine
stomach mucin (Nagata et al., 2005).
GFL exerted a strong cytotoxicity towards HeLa cells. The minimum GFL concentration
necessary to kill all the cells was 25 μg/ml (Kawagishi et al., 1990). Interestingly this toxicity
of GFL for HeLa cells was inhibited by preincubation of the lectin with its specific sugar
(GalNAc).
1.6.1.1.11. Inocybe umbrinella lectin
A lectin with a molecular weight of 17 kDa was extracted from the fruiting bodies of the toxic
mushroom I. umbrinella and named IUL (Zhao et al., 2009b). Several sugars could inhibit the
hemagglutinating activity of IUL such as raffinose, melibiose, lactose and galactose.
HIV-1 reverse transcriptase was inhibited by IUL with an IC50 of about 5 mM. Moreover,
IUL exhibited an antiproliferative effect towards hepatoma HepG2 and breast cancer MCF-7
cells. The IC50 values determined were 3.5 and 7.4 mM, respectively (Zhao et al., 2009b).
55. 35Chapter 1
1.6.1.1.12. Marasmius oreades lectin
A lectin called MOA was found in the fairy ring mushroom M. oreades. MOA has specificity
towards Galα1,3Gal/GalNAc. MOA is a heterodimeric protein of 50 kDa, with two subunits
of 33 and 23 kDa, respectively (Winter et al., 2002; Wohlschlager et al., 2011). MOA was
shown to possess a strong toxicity towards the nematode C. elegans and the amoeba A.
castellanii when both organisms were incubated in the presence of MOA-expressing E. coli.
Although all the C. elegans become L4 in the control treatment none of these worms reaches
the L4 stage when fed on MOA. In addition, MOA inhibited the growth of A. castellanii
(Wohlschlager et al., 2011).
1.6.1.1.13. Pholiota adiposa lectin
The P. adiposa lectin (PAL) is a homodimeric protein composed of two identical subunits of
16 kDa each. The plant polysaccharide inulin was the only carbohydrate compound which
inhibited the hemagglutinating activity of PAL (Zhang et al., 2009). The lectin induced strong
inhibitory activity against the cellular proliferation of HepG2 and MCF-7 tumor cells with an
IC50 value of 2.1 and 3.2 μM, respectively. Furthermore, PAL also potently inhibited the HIV-
1 reverse transcriptase with an IC50 value of 1.9 μM (Zhang et al., 2009).
1.6.1.1.14. Pleurotus citrinopileatus lectin
A 32.4 kDa lectin was extracted from fresh fruiting bodies of the edible mushroom P.
citrinopileatus. The hemagglutinating activity of PCL was inhibited be several sugars such as
maltose and insulin.
PCL showed potent antitumor effect in mice bearing sarcoma 180 with approximately 80%
inhibition of the tumor growth after 20 days treatment of the mice with PCL (5 mg/kg body).
Moreover the lectin exerted inhibitory activity against HIV-1 reverse transcriptase with an
IC50 of 0.93 μM (Li et al., 2008).
1.6.1.1.15. Pleurotus ostreatus lectin
POL is a melibiose-specific lectin isolated from the fruiting bodies of the oyster mushroom P.
ostreatus. The lectin is composed of two subunits with a molecular mass of 40 and 41 kDa,
respectively. Injection of POL into mice for 20 days at the dose of 1.5 mg/kg body weight
inhibited tumor growth of sarcoma S-180 and hepatoma H-22 cells by 88 and 75%,
respectively (Wang et al., 2000a).
56. 36Chapter 1
1.6.1.1.16. Schizophyllum commune lectin
A homodimeric lectin (SCL) was purified from the edible split gill mushroom S. commune.
The protein has a molecular mass of 64 kDa and is composed of two subunits of 32 kDa.
Lactose potently inhibited the activity of SCL (Han et al., 2005). SCL exerted a dramatic
inhibition against HIV-1 reverse transcriptase with an IC50 of 1.2 μM (Han et al., 2005).
Chumkhunthod et al. (2006) isolated another lectin from S. commune with a subunit of 31.5
kDa and different sugar specificity. This lectin showed high affinity towards GalNAc. The
GalNAc-specific SCL showed a potent cytotoxic effect towards human epidermoid carcinoma
cells with an IC50 value of 20 μg/ml (Chumkhunthod et al., 2006).
1.6.1.1.17. Tricholoma mongolicum lectin
Two lectins, named TML-1 and TML-2, have been purified from the mycelium of the edible
mushroom T. mongolicum. Both proteins are built up of two subunits with a similar molecular
weight of 17.5 kDa. The activity of TML1 and TML2 was abolished by several sugars such as
lactose, GalNAc and galactose (Wang et al., 1995). Both lectins exhibited antiproliferative
effects towards mouse monocyte-macrophage PU5-1.8 cells and mouse mastocytoma P815
cells. In addition, TML-1 and TML-2 inhibited the growth of sarcoma 180 cells by 69 % and
92%, respectively (Wang et al., 1996).
Feeding of the plant nematodes D. dipsaci and H. glycines on a diet containing TML-1 and
TML-2 revealed that both lectins possess nematotoxic activity (Zhao et al., 2009a). The effect
of TML-1 and TML-2 was time- and dose-dependent in both nematodes with LC50 values of
6.3 and >10 mg/ml, respectively, for D. dipsaci, while these values were 6.4 and 1.7 mg/ml,
respectively, for H. glycines.
Incubation of human hepatoma (H3B), human choriocarcinoma (JAr), mouse melanoma
(B16) and rat osteosarcoma (ROS) cell lines with TML-1 and TML-2 decreased the cell
viability in all cell lines as shown in Table 1.2 (Wang et al., 2000b).
57. 37Chapter 1
Table 1.2. Decrease in viability of different tumor cell lines after exposure to 1μM of TML-1 or TML-
2. The table was adapted from Wang et al. (2000b).
Tumor cell line % decrease in tumor cell viability
TML-1 (1μM) TML-2 (1μM)
H3B 58 ± 6 44 ± 3
B16 39 ± 3 56 ± 6
Jar 37 ± 2 26 ± 2
ROS 35 ± 1.4 41 ± 11
1.6.1.1.18. Volvariella volvacea lectin
The lectin VVL was isolated from the fruiting bodies as well as from cultured mycelia of the
edible mushroom, V. volvace. VVL is a homodimeric protein with a molecular mass of 32
kDa. The hemagglutinating activity of VVL was not inhibited by simple carbohydrates but it
was inhibited by thyroglobulin (She et al.,1998).
VVL was reported to exert a toxic effect towards mice with an LD50 of 17.5 mg/kg mice. In
addition, VVL showed antitumor activity against Sarcoma 180 cells. When mice were
inoculated with these tumor cells their lifespan was 12.5 ± 5 days but when these mice were
injected with 85 or 175 μg VVL per mouse the lifespan increased for 63 and 110 %,
respectively, which demonstrated the strong activity of VVL towards Sarcoma 180 cells (Lin
and Chou, 1984).
VVL exerted a strong reduction of the cell viability of T cells. The cell viability was reduced
by approximately 50% when the cells were incubated for 2h with 10 nM VVL and all the
cells died after 24 h incubation with 125 nM VVL (Ho et al., 2004).
1.6.1.2. Lectins from the fungal order Atheliales
1.6.1.2.1. Sclerotium rolfsii lectin
SRL is a lectin extracted from the sclerotial bodies of the soil-borne phytopathogenic fungus
S. rolfsii. The lectin was described as a homodimeric protein made up of two subunits of 17
kDa. SRL has high affinity towards Gal/GalNAc (Wu et al., 2001). SRL showed anti-
nematode activity against the common root knot nematode, Meloidogyne incognita.
Incubation of M. incognita juveniles with 47 μg/ml SRL for 48h resulted in 36 % mortality
58. 38Chapter 1
which increased to 48% with a 5-fold higher dose of SRL (Bhat et al., 2010). It was proposed
that binding of SRL to glycoproteins present on the digestive tract of the nematode might
explain the toxicity of SRL.
1.6.1.3. Lectins from the fungal order Boletales
1.6.1.3.1. Boletus edulis lectin
BEL was purified from fresh fruiting bodies of B. edulis. The lectin has specificity for
melibiose and xylose. It is a homodimeric lectin that is built of two subunits of 16.3 kDa
(Zheng et al., 2007).
BEL exerted anti-nematode activity towards D. dipsaci and H. glycines (both plant parasitic
nematodes). For example feeding of both nematodes on a diet containing 10 mg/ml BEL for
48h resulted in 34 and 59% mortality, respectively (Zhao et al., 2009a).
BEL also showed an inhibitory effect towards human immunodeficiency virus-1 reverse
transcriptase with an IC50 of 14.3 μM (Zheng et al., 2007).
Furthermore BEL was reported to inhibit the proliferation of human carcinoma cell lines
dramatically. The proliferation of the colon cancer cells HT29 was inhibited for 92% at a
concentration of 10 µg/ml BEL. Less inhibition was observed in liver cancer cells (HepG2)
and breast cancer cells (MCF-7) with 79% and 77% inhibition, respectively, at the same
concentration (Bovi et al., 2011).
1.6.1.3.2. Boletus venenatus lectin
The BVLs are a family of isolectins that were purified from the mushroom B. venenatus and
were named BVL-1 to -8, respectively. All BVLs have a similar molecular weight (33 kDa)
and are composed of three identical subunits of 11 kDa. Mono- and oligosaccharides failed to
inhibit BVL activity, but the lectin activity was strongly inhibited by glycoproteins such as
asialofetuin (Horibe et al., 2010). BVLs exert high toxicity towards mice and rats. Injection of
BVLs into mice at a ratio of 0.5 mg/mouse resulted in killing of all the mice within a day after
the injection. Moreover, although oral feeding of rats on a diet containing 40 mg BVLs/kg
body did not kill these animals, they suffered from diarrhea about 4h after lectin application.
Interestingly, using an anti-diarrheal before BVL treatment prevented the rats to suffer from
diarrhea (Horibe et al., 2010).
59. 39Chapter 1
1.6.1.3.3. Xerocomus chrysenteron lectin
XCL is a lectin identified from the edible mushroom X. chrysenteron. The lectin is specific
for GalNAc and Gal and has a molecular weight of 15 kDa (Trigueros et al., 2003). XCL
exerted toxic effects on fruit fly, D. melanogaster and pea aphid, Acyrthosiphon pisum with
an LC50 of 0.4 and 0.7 mg/ml, respectively (Trigueros et al., 2003). XCL was shown to be
internalized in insect (SF9) or mammalian (NIH-3T3 and Hela) cell lines via a clathrin-
dependent pathway (Francis et al., 2003). Moreover, feeding of Myzus persicae nymphs on an
artificial diet containing different concentrations of XCL for 24h resulted in a significant
mortality of the insects with an LC50 of 0.46 mg/ml. In addition the lectin also exerted toxic
effects on other biological parameters such as development time, weight and fecundity
(Karimi et al., 2007).
A recent report showed that XCL has a highly significant effect on the growth of the
nematode C. elegans and the mosquito A. aegypti (Bleuler-Martínez et al., 2011). At the time
when all the larvae of C. elegans and A. aegypti reached L4 and L2, respectively, in the
control treatment, 0 and 6% of the respective larvae fed on E. coli cells expressing XCL
reached the same stage.
XCL also caused a dose-dependent inhibition of cellular proliferation of two mammalian cell
lines, namely Hela and NIH-3T3 cells (Marty-Detraves et al., 2004), and it was proposed that
XCL interferes with the cell adhesion process by binding to receptors on the cell surface.
1.6.1.4. Lectins from the fungal order Cantharellales
1.6.1.4.1. Rhizoctonia bataticola lectin
RBL is a lectin isolated from the mycelium of the phytopathogenic fungus R. bataticola. The
lectin shows high affinity towards complex sugars (Nagre et al., 2010). The molecular mass
of RBL is about 44 kDa and the protein consists of four subunits of 11 kDa.
RBL exerted a significant cytotoxic effect on the human ovarian cancer cell line PA-1 in a
concentration-dependent manner with an LC50 of 0.15 µM (Nagre et al., 2010).
1.6.1.4.2. Rhizoctonia solani agglutinin
R. solani agglutinin, known as RSA, is a lectin that was purified from the soil pathogen R.
solani (Vranken et al., 1987). RSA was found to be a homodimeric protein consisting of two
identical subunits of 15.5 kDa. The lectin has high affinity for GalNAc/Gal and more complex
60. 40Chapter 1
glycoproteins (Candy et al., 2001). RSA is structurally and evolutionary related to the family
of proteins possessing a ricin-type lectin motif (Candy et al., 2001). R. solani produces black
sclerotia in harsh conditions. Since RSA is an abundant protein in these sclerotia the lectin
was proposed to play role as a storage protein (Kellens and Peumans, 1990).
1.6.1.5. Lectins from the fungal order Polyporales
1.6.1.5.1. Ganoderma capense lectin
GCL is a lectin isolated from the medicinal mushroom G. capense. The lectin has a molecular
mass of 18 kDa and its activity can be inhibited by Gal/GalNAc (Ngai and Ng, 2004). GCL
induced proliferation inhibitory activity against three cancer cell lines L1210, M1 and HepG2
with IC50 values of 8 μM, 12.5 μM and 16.5 μM, respectively (Ngai and Ng, 2004).
1.6.1.5.2. Ganoderma lucidum lectin
GLL is a lectin isolated from the fruiting bodies of the mushroom G. lucidum. The lectin was
found to be a hexameric protein with subunits of 18.5 kDa. Simple sugars failed to inhibit the
hemagglutinating activity of GLL which was inhibited by glycoproteins such as fetuin and
fibrinogen (Thakur et al., 2007). A different lectin was purified from G. lucidum and was
found to be a monomer with a molecular mass of 15 kDa (Girjal et al., 2011)
A strong toxicity (LC50=1.7 mg/ml) of GLL was induced when the plant nematode H.
glycines was fed on GLL for 48h. A lower toxicity was observed with the nematode D.
dipsaci. When this worm was fed on diet containing 10 mg/ml GLL for 48h the mortality rate
was about 34% (Zhao et al., 2009a).
Interestingly, GLL possesses a significant antifungal effect towards several phytopathogens
and dermatophytic fungi. The activity was determined as the Minimum Inhibitory
Concentration (MIC) of GLL against different fungi (Table 1.3) (Girjal et al., 2011). To our
knowledge no fungal lectins with antifungal activity except for GLL have been reported. So
far only very few plant lectins with antifungal activity have been reported, such as the potato
tuber lectin, the stinging nettle lectin, the wheat germ lectin and the flageolet bean lectin
(Broekaert et al., 1989;Gozia et al., 1995; Ciopraga et al., 1999; Xia and Ng, 2005).
61. 41Chapter 1
Table 1.3. Minimum Inhibitory Concentration (MIC) of GLL against pathogenic fungi causing plant
diseases (Phytopathogens) and skin diseases (Dermatophytes). The table was adapted from (Girjal et
al., 2011).
Phytopathogenic fungi Dermatophytic fungi
Fungal strains MIC
( μg/ml)
Fungal strains MIC
( μg/ml)
Fusarium oxysporum 20 Trichophyton rubrum 65
Penicillium chrysogenum 35 Trichophyton tonsurans 20
Aspergillus niger 50 Trichophyton interdigitale 20
Colletotrichum musae 60 Epidermophyton floccosum 15
Botrytis cinerea 65 Microsporum canis 70
1.6.1.6. Lectins from the fungal order Russulales
1.6.1.6.1. Hericium erinaceum lectin
The H. erinaceum agglutinin (HEA) was extracted from the fruiting bodies of the monkey
head mushroom H. erinaceum. The lectin has a molecular mass of 51 kDa and has high
affinity towards inulin (Li et al., 2010). HEA exhibited potent inhibition of the cellular
proliferation of hepatoma (HepG2) and breast cancer (MCF-7) cells with IC50 values of
56 and 77 μM, respectively. The lectin also exerted high inhibition activity against HIV-1
reverse transcriptase with an IC50 of 32 μM.
1.6.1.6.2. Russula delica lectin
RDL is a dimeric lectin found in the fresh fruiting bodies of the mushroom R. delica. The
lectin consists of two identical subunits of 30 kDa. RDL showed high specificity towards
inulin and o-nitrophenyl-β-D-galactopyranoside (Zhao et al., 2010). RDL manifested high
HIV-1 reverse transcriptase inhibitory activity with an IC50 of 0.26 μM. Furthermore, the
proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells was strongly inhibited
by RDL with IC50 values of 0.52 and 0.88 µM, respectively.
1.6.1.6.3. Russula lepida lectin
The R. lepida lectin (RLL) was isolated from dried fruiting bodies of the mushroom R. lepida.
The lectin is composed of two subunits with a mass of 16 kDa each. Inulin and O-
62. 42Chapter 1
nitrophenyl-β-D-galacto-pyranoside inhibited the hemagglutinating activity of RLL. In
addition, RLL demonstrated antiproliferative activity towards two tumor cell lines, MCF-7
and Hep G2 with IC50 values of 0.9 and 1.6 mM, respectively (Zhang et al., 2010b).
1.6.1.7. Lectins from the fungal order Thelephorales
1.6.1.7.1. Boletopsis leucomelas lectin
KL-15 is a lectin isolated from the edible mushroom Kurokawa (B. leucomelas). The lectin
consists of a single polypeptide of 15 kDa (Koyama et al., 2002). The cellular proliferation of
human monoblastic leukemia U937 was inhibited by KL-15 in a dose-dependent manner with
an IC50 of approximately 15 mg/ml. The effect of KL-15 in U937 cells was apoptosis-
dependent as was clearly determined via observation of typical apoptosis features such as
formation of apoptotic bodies, nuclear condensation, and DNA fragmentation (Koyama et al.,
2002).
1.6.2. Ascomycota
1.6.2.1. Lectins from the fungal order Eurotiales
1.6.2.1.1. Paecilomyces japonica lectin
PJL is a sialic acid-specific lectin that was extracted from the mushroom P. japonica. The
molecular mass of PJL is 16 kDa (Park et al., 2004). PJA decreased the cell viability of
human stomach cancer SNU-1 cells, human pancreas cancer AsPc-1 cells, and human breast
cancer MDA-MB-231 cells by 65, 46 and 30%, respectively, when PJA was dosed at 1 μM.
In contrast only a small effect was observed on human colon cancer SNU-C1cells, human
lung cancer A549 cells, human bladder cancer T24 cells, and human liver cancer Hep3B cells
with toxicity about 7 ± 2% in all cell lines (Park et al., 2004).
1.6.2.1.2. Penicillium chrysogenum lectin
The P. chrysogenum lectin, abbreviated as PeCL, is a lectin produced in the mycelium of the
fungus P. chrysogenum. The activity of PeCL was counteracted by mannose and the lectin has
a molecular mass of 71 kDa divided on two subunits of 31 and 40 kDa, respectively (Francis
et al., 2011).
PeCL exerted significant differences in mortality of the green peach aphid, M. persicae when
this aphid was fed on an artificial diet containing PeCL with an LC50 value of 0.4 mg/ml
