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Protein J (2012) 31:439446

DOI 10.1007/s10930-012-9423-8

Receptors of Garlic (Allium sativum) Lectins and Their Role


in Insecticidal Action
Santosh K. Upadhyay Pradhyumna K. Singh

Published online: 24 May 2012


Springer Science+Business Media, LLC 2012

Abstract Garlic (Allium sativum) lectins are promising ASAL Allium sativum leaf agglutinin
candidate molecules for the protection against chewing GNA Galanthus nivalis agglutinin
(lepidopteran) as well as sap sucking (homopteran) insect BBMV Brush border membrane vesicles
pests. Molecular mechanism of toxicity and interaction of
lectins with midgut receptor proteins has been described in
many reports. Lectins show its effect right from sensory 1 Introduction
receptors of mouth parts by disrupting the membrane
integrity and food detection ability. Subsequently, enter Insecticidal d-endotoxins of Bacillus thuringiensis (Bt),
into the gut lumen and interact with midgut glycosylated plant derived defence related proteins and siRNA molecules
proteins like alkaline phosphatase (ALP), aminopeptidase- provide protection against insect pests and are alternatives to
N (APN), cadherin-like proteins, polycalins, sucrase, chemical pesticides [4, 64, 81, 83, 86]. Lectins are one of the
symbionin and others. These proteins play critical role in plant derived insecticidal proteins. These are also known as
life cycle of insect directly or indirectly. Lectins interfere agglutinins. Lectins contain at least one non-catalytic
with the activity of these proteins and causes physiological domain which binds reversibly to specific monosaccharides
disorders leading to the death of insects. Lectins further or oligosaccharides. Lectins are isolated from various plant
transported across the insect gut, accumulated in various tissues and other organisms with specific carbohydrate
body parts (like haemolymph and ovary) and interact with specificity. They have been categorized on the basis of sugar
intracellular proteins like symbionin and cytochrome p450. specificity [27, 32, 40, 65, 71, 80, 86, 92, 94, 97].
Binding with cytochrome p450 (which involve in ecdysone Among the plant derived lectins, mannose-binding lec-
synthesis) might interfere in the development of insects, tins isolated from garlic bulb [95] and leaf [81] have gained
which results in growth retardation and pre-mature death. attention in recent decades. These are toxic to both chew-
ing (lepidopteran) and sap-sucking (homopteran) type
Keywords Garlic lectins  Glycosylated  Homopteran  insect pests and considered to be safe in principle due to the
Insect  Lepidopteran edible source of origin [5, 8, 23, 24, 59, 77, 78, 81]. Garlic
lectins also show rapid agglutination of rabbit erythrocytes
Abbreviations [8, 81] and inhibitory effect against HIV1 and HIV2
ALP Alkaline Phosphatase induced cytopathicity in MT-4 cells [8].
APN Aminopeptidase-N
ASA Allium sativum agglutinin
2 Structure of Garlic Lectins

S. K. Upadhyay  P. K. Singh (&) Garlic lectins are dimeric proteins. Garlic bulb lectins are
CSIR-National Botanical Research Institute, Council
either heterodimer (ASAI) of *11.5 and *12.5 kDa subunits
of Scientific and Industrial Research, Rana Pratap Marg,
Lucknow, UP 226001, India or homodimer (ASAII) of *12 kDa subunits [95]. However,
e-mail: pradhyumnasingh@hotmail.com garlic leaf lectin is a homodimer of *12 kDa subunits. These

123
440 S. K. Upadhyay, P. K. Singh

Dysdercus
cingulatus Spodoptera litura
Aphis gossypii Helicoverpa armigera

Nephottix

Acyrthosiphon
pisum
Nilaparvata lugens

Allium sativum agglutinin

Sogatella furcifera
Myzus persicae

Aphis craccivora
Myzus nicotianae
Tetranychus kanzawai Lypaphis erysimi

Fig. 1 Insects, which are sensitive to garlic lectins. Bioassay was performed with protein preparations from native (garlic bulb and leaf) and
recombinant source (expressed in E. coli, yeast and transgenic plants)

are structurally and evolutionarily related to Galanthus nivalis [16, 48, 55, 62, 78, 88]. These are toxic to a number of
agglutinin (GNA) [81, 95]. Primary structures of both bulb insect pests of order lepidoptera and homoptera [8, 77, 78,
and leaf lectins of garlic are more than 80 % similar [15, 81]. 81]. The insecticidal properties of garlic lectins have been
Crystal structure of dimeric garlic bulb lectin shows b-prism II explored by the protein preparations from native source
fold structure, similar to GNA. It contains three 4-stranded (i.e. bulb and leaves), recombinant source (i.e. E. coli and
anti-parallel b-sheets, arranged on three surfaces of a trian- yeast) and in transgenic plants [29, 30, 77, 85]. Several
gular prism, forming a 12-stranded b-barrel and inter-related transgenic plants expressing garlic lectin have shown
by an approximate threefold internal symmetry. This is con- resistance against different insect pests (Fig. 1, Table 1).
sistent with the tandem sequence repeats in primary structure, Transgenic tobacco expressing garlic lectin offers resis-
suggesting domain triplication in the gene during evolution tance against Spodoptera litura [77] and Myzus persicae
[15]. Unlike the snowdrop lectin which exists as a tetramer, [23], transgenic rice against brown plant hopper, green
garlic lectins always exist as dimer, despite of high degree of leafhopper and white backed plant hopper [10, 105] and
sequence similarity between them. Each subunit of garlic transgenic chickpea against Aphis craccivora [14].
lectins contains three highly conserved mannose binding sites, Although, the potential of garlic lectins for the insect
QXDXNXVXY [15, 6870]. control has been established, d-endotoxins of Bt are more
toxic against lepidopteran pests. Nevertheless, none of the
d-endotoxins have shown insecticidal activity against
3 Garlic Lectins as Bio-pesticides homopteran pests [22, 99]. Therefore, pest control strate-
gies in future would be based on the pyramiding of two
Lectins are one of the most promising family of proteins toxins for the effective control of both types of insects. It
for the development of insect resistant transgenic plants has been demonstrated that garlic leaf lectin and Cry1Ac

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Receptors of Garlic 441

Table 1 Insecticidal activity of garlic lectins Table 2 Putative receptors of garlic lectins in midgut of different
insects
Insect Method of toxicity References
assay Insect Size of Identification References
putative
Helicoverpa armigera Synthetic diet [5, 85] receptor
Spodoptera littoralis Transgenic tobacco [77, 85] (kDa)
Nilaparvata lugens Transgenic rice [10, 105]
Helicoverpa 113 Midgut [84]
(brown plant hopper)
armigera aminopeptidase
Nephotettix spp. Synthetic died [54] APN2
(green leafhopper)
Helicoverpa 104 AGAP009726-PA, [84]
Nephotettix spp. Transgenic rice [10, 105] armigera partial (Cadherin-N
(green leafhopper) protein)
Sogatella furcifera Transgenic rice [10, 105] Helicoverpa 102 Polycalin [84]
(White backed plant armigera
hopper)
Helicoverpa 82 Polycalin [84]
Lypaphis erysimi Synthetic diet [8, 54] armigera
(Aphid)
Helicoverpa 59 Alkaline phosphatase [84]
Lipaphis erysimi Transgenic Indian [24] armigera 2
mustard
Helicoverpa 59 Alkaline phosphatase [84]
Myzus persicae Transgenic tobacco [23] armigera 1
(peach potato Aphid)
Helicoverpa 57 Cytochrome P450 [84]
Myzus nicotianae Transgenic Arabidopsis [77] armigera CYP315A1
(Tobacco aphid) thaliana
Acyrthosiphon 110 Alanyl [30]
Aphis craccivora Synthetic died [54] pisum aminopeptidase N
(cow-Pea aphid)
Acyrthosiphon 66 Sucrase [30]
Aphis craccivora Transgenic chickpea [14] pisum
(cow-Pea aphid)
Lipaphis 57 Symbionin from [9]
Dysdercus cingulatus Synthetic diet [8] erysimi Buchnera
(red cotton bug) aphidicola
Acyrthosiphon pisum Synthetic diet [30] (Q93T48)
(pea aphid) Lypaphis 55 NI [8]
erysimi
Dysdercus sp. 45 NI [8]
Aphis 66 NI [54]
d-endotoxins can be used together without interfering the craccivora
activity of each other [88]. Nephotettix Four proteins NI [78]
virescens from 30 to
66 kDa
M. persicae 40, 66 and NI [23]
4 Putative Receptors of Garlic Lectins in the Midgut 70 kDa
of Insects NI not identified

Binding of toxins to receptors in the insect gut is an


important step in toxicity. This also determines the speci- 4.1 Cadherin like Protein
ficity of a toxin to insect. Garlic lectins bind to several
proteins (cadherin-like proteins, glycosyl phosphatidyl- Cadherins belong to diverse superfamily of proteins with a
inositol (GPI)-anchored aminopeptidase-N (APN), GPI- variety of functions, like cell adhesion, migration, cyto-
anchored alkaline phosphatase (ALP), polycalins, sucrase, skeletal organization and morphogenesis [3, 36]. Cadherins
cytochrome P450, symbionin and others) in the midgut generally possess 534 calcium binding domains or cad-
of insects (Table 2) through glycan mediated interactions herin repeats [3, 21, 61] and/or laminin, epidermal growth
[9, 54, 84]. Most of these receptor proteins are membrane factor-like repeats [60] and mucin domains [34]. Expres-
bound enzymes. Some of them like ALP, APN and poly- sion of cadherins is highly regulated temporally as well as
calins are earlier reported as receptors of d-endotoxins. spatially and sometimes limited or unique to a particular
This is due to the lectin like structure of third domain of d- cell type. These are glycosylated proteins and usually
endotoxins, which binds to specific sugar residues on anchored on membranes by single trans-membrane
receptors [12, 79]. domain. But cadherins with seven-trans-membrane

123
442 S. K. Upadhyay, P. K. Singh

domains [89] or GPI-anchor has also been reported [98]. 4.3 GPI-Anchored Alkaline Phosphatase (ALP)
Cadherins are primarily characterized as receptor of
d-endotoxins [35, 43, 90] which consisted of a signal ALPs are also a family of proteins and function as a
peptide, 12 cadherin repeats, a membrane proximal extra- receptor of d-endotoxins in H. armigera [87], M. sexta
cellular domain, a transmembrane domain and a small [58], H. virescens [45, 46, 51] and others. Glycan mediated
cytoplasmic domain [91]. Similar domain organization has binding of ALPs to d-endotoxins has been demonstrated
been reported for cadherin receptors of d-endotoxins in [79]. Garlic leaf lectin also binds to ALP of H. armigera
insects [64]. However, garlic leaf lectin interact wih BM1 [84] and inhibits the phophatase activity like Cry1Ac
type of cadherin in H. armigera [84] which is different [79, 88]. Inhibition in enzyme activity might play some
from the d-endotoxin specific cadherins. BM1 cadherin role in insecticidal activity. Like APNs, ALPs are also
consists of additional leminin G and EGF domains with expected to involve in internalization of lectin. Interference
cadherin repeats. in ALP activity in haemolymph or other tissue might also
contributed in toxicity.

4.2 Glycosylphosphatidyl-Inositol (GPI)-Anchored 4.4 Other Proteins


Aminopeptidase-N (APN)
Garlic leaf lectin also interacts with polycalins which
APNs are also a family of proteins which cleave peptide consist of multiple lipocalin domains [84]. Mauchamp
bonds from the N-terminus of proteins to release amino et al. [57] isolated this protein for the first time from
acids. These are known for variety of functions in different Bombyx mori but now it is reported in other insects like H.
species and work along with endopeptidases and carb- armigera also [2, 56]. Alternative splicing in the transcript
oxypeptidases for proteins digestion in insect gut [101]. generates different form of polycalins which enhances their
These belong to the gluc-zincins subfamily of zinc binding diversity and binding specificities. These proteins generally
metalloprotease/peptidase superfamily [41]. Enzymes of consist of signal peptide, GPI anchor attachment and sev-
this family contain a zincin motif (HEXXH; where X can eral potential sites for glycosylation. Some of the sequen-
be any amino acid) and a conserved glutamic acid residue ces of polycalins match with d-endotoxin binding proteins
at 24th position downstream of the first histidine. First [42, 64]. Polycalins have been reported as receptor of
glutamic acid residue is important for enzyme catalysis, Cry1Ac in H. armigera [2, 56].
while histidines and last glutamic acid residue serve as zinc Fitches et al. [30] reported the interaction of garlic bulb
ligands. The enzyme also consists of another highly con- lectin with sucrase in gut of aphid A. pisum. Gut sucrase is
served GAMEN motif which forms the part of active site involved in carbon nutrition and osmoregulation. It is
[52]. APNs are about one thousand amino acid residues localized in the aphids distal midgut and mediates the
long proteins and post translational modification in various hydrolysis of sucrose into glucose and fructose [6, 17].
ways produces 70170 kDa mature proteins. Several forms Most of the fructose and some glucose utilized in respira-
of APNs have been isolated and characterized. Presence of tion and anabolism [6]. Remaining glucose used in trans-
N- or O-linked glycosylation has also been reported [2, 12, glucosidation via a gut transglucosidase activity and
49]. N-terminal signal peptide directs APNs to the outer incorporated into glucose-dominated oligosaccharides of
surface of the cytoplasmic membrane where they bind to 20 hexose units, which later on annulled in the aphids
the membrane by GPI anchor [19, 50, 53, 82]. Angelucci honeydews [6, 76, 100]. The transglucosidation of glucose
et al. [2] have reported seven APN genes in H. armigera, reduces the osmotic pressure of the gut contents and hon-
four among them interact with Cry1Ac d-endotoxin. It is eydew becomes isosmotic to the aphid body fluids [20, 26,
noteworthy that APNs have been comprehensively studied 103]. A chemical inhibitor of sucrase decreases the mean
as receptors of d-endotoxins [2, 44, 49, 87]. aphid survival to 2.8 days [47]. Binding of lectins might
Fitches et al. [30] observed the interaction of garlic bulb interfere in sucrase activity, which results in toxicity. The
lectin with the membrane bound alanyl aminopeptidase N observation supports the acute toxicity of lectins to insects
of Acyrthosiphon pisum. APN2 of H. armigera interacts at higher doses [29, 30].
with garlic leaf lectin [84] which is well known receptor of Garlic leaf lectin also interacts with Cytochrome P450
d-endotoxins. It is suspected that lectins inhibit the APN (CYP315A1) in H. armigera [84]. Cytochrome P450 is a
activity, which results in toxicity. However, we observed member of highly conserved halloween gene family in
that the garlic leaf lectin does not inhibit the APN activity insects and involved in the biosynthesis of the insect
[88]. Since the exact mechanism is not known, APNs are molting hormone, 20-hydroxyecdysone (20E) [33, 7274,
expected to involve in the transport of garlic lectins to the 102]. Ecdysones play major role in life cycle (embryonic,
haemolymph, as observed for GNA [67]. larval, pupal and adult development and reproduction) of

123
Receptors of Garlic 443

insects [75]. Cytochrome P450 is a cytosolic protein and exhibiting similar glycan specificity; might have different
therefore cannot function as an extracellular receptor. It is effect on closely related insects [13]. Similarly, one lectin
possible that lectins first accumulate into the haemolymph might cause different effect on closely related insects.
and then interact with Cytochrome P450. This results delay Garlic lectins show the glycan mediated interaction with
in larval development and premature death of insect. several putative receptors in the midgut of insects [9, 54,
Severe growth retardation has been reported in several 84]. Interactions with the digestive and moulting enzymes
insects after lectin ingestion [85]. are also carbohydrate mediated [30, 84]. This shows that
Banerjee et al. [9] reported symbionin (SymL) in Lipaphis carbohydrate affinity of lectin plays critical role not only in
erysimi as a receptor of garlic leaf lectin. Buchnera aphidi- other known biological functions but also in the insect
cola, a bacterial endosymbiont of aphid encodes this protein specificity and insecticidal activity [13, 96].
[1, 25, 106]. It is a glycoprotein and its carbohydrate moiety
plays critical role in binding to the lectin [9]. Symbionin is
found in haemolymph only and therefore cannot function as 6 Mode of Insecticidal Action
receptor. Antibiotic treatment of pea aphids reduced the
amount of symbionin in heamolymph but do not change Study on the mechanism of insecticidal action of lectins
sensitivity of the insect with garlic lectins [30]. Rice brown was initiated nearly 25 year ago. Gatehouse et al. [31]
plant hopper maintains yeast as its endosymbiont which does proposed that lectins inhibit the nutrient absorption or
not produce symbionin and yet sensitive to garlic lectins cause severe disruption of the epithelial cells in midgut by
[66]. These studies establish that the interaction with sym- stimulating endocytosis. This results in disorganization and
bionin is not necessary for the insecticidal activity of garlic elongation of the striated brush border microvilli which
lectins, this might play some other role. leads to the swelling of epithelial cells and complete clo-
Several other proteins of various sizes have been sure of the gut lumen [28, 37, 38, 67]. Powell et al. [67]
reported from different insects, which show interaction reported the transportation of lectin across the midgut
with garlic lectins [8, 23, 78]. In-depth studies are required epithelial barrier after binding to the midgut glycoproteins
to identify and characterize such proteins and elucidate and disruption in the microvilli of striated brush border
their contribution in the toxicity of garlic lectins to insects. region. Sauvion et al. [80] reported the interaction of a
lectin with glycosylated receptors on the surface of stom-
ach epithelial cells which cause interference in normal
5 Role of Glycans in Receptors-Lectins Interaction metabolism and cell functions leading to the rapid feedback
and Insecticidal Action response on feeding behaviour. These results suggest that
each lectin has different mode of action for different insects
Lectins interact non-covalently with carbohydrate moieties at the cellular level. Insecticidal actions of garlic lectins to
of glycosylated proteins and display high affinity and homopteran and lepidopteran insects have been described
specificity for their ligands. Glycan binding ability of lec- in detail (Fig. 2).
tins has been established by several methods like inhibition
of agglutination, enzyme-linked lectin adsorbent assay, 6.1 Insecticidal Action Against Lepidopteran Insects
surface plasmon resonance (SPR), isothermal titration
calorimetry and glycan array [7, 11, 18, 85]. X-ray dif- A hypothetical mode of insecticidal action of garlic lectins
fraction experiments, used for the three-dimensional against lepidopteran insects is described (Fig 2a).
structures analysis of several plant lectins, have also con- According to published literature and our own observation,
tributed in the study [15]. Co-crystallization of lectins has the garlic lectins interact with several glycosylated BBMV
given the information about their interactions with simple proteins like Cadherin, APN, ALP, polycalins and others in
and complex carbohydrate moities and enlightened the the midgut peritropic membrane [84]. Since most of them
amino acids residues involved in the interaction. Lectins are reported as receptor for d-endotoxins, there is possi-
have been classified as glucose/mannose, N-acetylgluco- bility that a few or all functions as receptor for garlic
samine, galactose/N-acetylgalactosamine and fucose-bind- lectins also. Binding of lectins to the midgut proteins affect
ing, according to their specificity. Van Damme et al. [93] the nutrient absorption. This might be due to the inhibition
have constituted a new group of lectins which are isolated of digestive and other related enzymes. Further, garlic
from monocotyledonous plants and show very high man- lectins are resistant to the midgut proteases and transported
nose specificity. Lectins interact with the insects intestinal to haemolymph. Where, these lectins interact with a
epithelial cells or peritrophic membrane and/or glycosyl- Cytochrome P450 and interfere in larval development. This
ated digestive enzymes [30, 63, 84]. However, the exact results in severe growth retardation and premature death
mechanism of insecticidal action is unknown. Lectins [84].

123
444 S. K. Upadhyay, P. K. Singh

(A) Ingestion of garlic (Allium (B) Ingestion of garlic (Allium


sativum) lectins sativum) lectins

Glycan mediated interaction of garlic lectins to Carbohydrate mediated binding of Carbohydrate mediated binding of
the midgut brush border membrane vesicle (BBMV) garlic lectins to the mouth parts garlic lectins to the proteins like
proteins [as cadherin like proteins, aminopeptidases sensory receptor of insects aminopeptidases (APN), sucrase
(APN), alkaline phosphatases (ALP), Polycalins and and others in midgut epithelial
others] cells of insects
Disruption in membrane integrity
and food detection ability of
insects Inhibition in nutrient absorption and alteration
Inhibition in nutrient absorption and alteration in the enzyme activity of APN and sucrase,
in the enzyme activity of ALP and APN which leads to the physiological disorders
leads to physiological disorders. Death of insects due to starvation

Inhibition in sucrase Internalization of lectins


activity might cause in haemolymph shows
Internalization of garlic lectins in osmotic imbalance stability against gut
haemolymph, show stability against gut between gut and proteases
proteases haemolymph

Interaction with proteins


Interaction of garlic lectins with Cyt p450 in midgut Transfer of body fluid to like symbionin and
microsomes or fat body and might interfere in molting gut and insects appear others
and development of insects. These might also inhibit the shrivel and finally dead
ALP activity in haemolymph and other tissue

Death of insects by
unknown mechanism
Severe growth retardation and/or death of
insects by unknown mechanism

Fig. 2 a A hypothetical mechanism of insecticidal action of garlic insecticidal action of garlic lectin against Homopteran insects,
lectins against lepidopteran insects, summarized from the several summarized from the several studies in different experimental
studies in different experimental conditions reported in literature conditions reported in literature (cited in the text and tables)
(cited in the text and tables). b A hypothetical mechanism of

6.2 Insecticidal Action Against Homopteran Insects lepidopteran insect pests. Due to the edible source of origin
garlic lectins are supposed to be biosafe. The insecticidal
Insecticidal action of garlic lectins against homopteran action of garlic lectins is a multi-step process that involves
insects slightly differ from the lepidopteran insects the interaction with several glycosylated receptor proteins in
(Fig. 2b). Garlic lectins bind to the sensory receptors of midgut of insects. This results in inhibition in nutrient
mouth parts which reduce the food detection ability of absorption leading to mortality. Garlic lectins get accumu-
insects resulting in starvation and eventually death [39, 80]. lated into the haemolymph and ovarioles and interfere in the
Subsequently, lectins enter into the insect gut and interact development and reproduction. Although, several reports on
with glycosylated gut proteins like APN, sucrase and others the receptors of garlic lectins have been summarised in this
in midgut epithelial cells [30]. These result in inhibition in review, still detail study on their interaction with lectins and
nutrient absorption. Further, garlic lectins might alter the post interaction processes will be imperative in order to
enzyme activity of APN and sucrase, which ultimately understand the insecticidal mechanism. In addition, the
disturb the physiology of insects. Inhibition of sucrase identification of binding epitopes will be helpful in engi-
activity has been correlated with the osmotic imbalance in neering the lectins for more specificity, higher toxicity and
aphid gut leading to insect mortality [30, 47]. Similar to delay the resistance development in insects.
lepidopteran insects, garlic lectins get accumulated in the
haemolymph and ovarioles [29]. Accumulation in ovarioles Acknowledgment Authors are grateful to the Council of Scientific
and Industrial Research, Government of India for funding the
might be responsible for the loss in fecundity. The role of
research. SKU is thankful to CSIR for senior research fellowship and
garlic lectins in the haemolymph and mechanism of tox- G B Technical University, Lucknow, UP, India for Ph.D. registration.
icity is need be elucidated in detail in future.

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