Structural aspects and biomedical applications of microfungal lectins

International Journal of Biological Macromolecules 134 (2019) 1097–1107

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Review

Structural aspects and biomedical applications of microfungal lectins Ram Sarup Singh a,⁎, Amandeep Kaur Walia a, John F. Kennedy b a b

Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala 147 002, Punjab, India Chembiotech Laboratories, Advanced Science and Technology Institute, 5 The Croft, Buntsford Drive, Stoke Heath, Bromsgrove, Worcs B604JE, UK

a r t i c l e

i n f o

Article history: Received 11 March 2019 Received in revised form 15 May 2019 Accepted 15 May 2019 Available online 16 May 2019 Keywords: Microfungi Lectins Glycoproteins Anti-cancer Immunomodulatory

a b s t r a c t Lectins are unique biorecognition molecules that identify cell surface carbohydrates/glycoproteins and play significant role in various interactions. They are ubiquitous glycan-specific proteins/glycoproteins of non-immune origin. Amongst microfungi, lectins have been widely reported from aspergilli, penicilli, Fusarium sp., etc., however a plethora of genera still remains unexplored. Microfungal lectins have wide diversity in their haemagglutination and carbohydrate specificity. They also exhibit great variations in their structural organization which influences lectin-glycan interactions. The present review summarizes the sources, characteristics and structural diversity of microfungal lectins. Prospective biomedical applications of microfungal lectins as anticancer, mitogenic, immunomodulatory, antioxidant and therapeutic agents have been discussed extensively. © 2019 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of microfungal lectins and their haemagglutination activity . Characteristics of microfungal lectins . . . . . . . . . . . . . . . Structural aspects of microfungal lectins . . . . . . . . . . . . . 4.1. Three dimensional (3D) structure of microfungal lectins . . . 4.1.1. Fungal fucose-specific lectin family . . . . . . . . 4.1.2. Ricin B-chain like family (R-type) . . . . . . . . . 4.1.3. Fungal fruit body (ABL-like) family . . . . . . . . 5. Biomedical applications of microfungal lectins . . . . . . . . . . . 5.1. Cytotoxic activity of microfungal lectins on cancerous cells . . 5.2. Effect of microfungal lectins on proliferation of immune cells . 5.3. Immune stimulating effect of microfungal lectins . . . . . . 5.4. Therapeutic potential of microfungal lectins . . . . . . . . 5.5. Antimicrobial activity of microfungal lectins . . . . . . . . 5.5.1. Antibacterial activity . . . . . . . . . . . . . . . 5.5.2. Antifungal activity . . . . . . . . . . . . . . . . 5.6. Antioxidant activity of microfungal lectins . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

⁎ Corresponding author. E-mail address: [email protected] (R.S. Singh).

https://doi.org/10.1016/j.ijbiomac.2019.05.093 0141-8130/© 2019 Elsevier B.V. All rights reserved.

Lectins are heterogenous group of proteins of non-immune origin which interact with glycoproteins on cell surface, in cytoplasmic or nuclear structures and extracellular matrix [1]. They interact noncovalently with cell surface carbohydrate moieties/glycoconjugates

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R.S. Singh et al. / International Journal of Biological Macromolecules 134 (2019) 1097–1107

through molecular sites, with high affinity and specificity [2]. The spatial arrangement of amino acids constituting the carbohydrate recognition domain (CRD) and neighbouring amino acids determines the specificity of the carbohydrate-binding site [3]. Owing to their specificity, lectins have broad implication as mediators during various host-pathogen interactions [4–6]. They act as biorecognition molecules and play significant role in cell-cell interactions by identification of carbohydrate moieties in cell surface and glycoconjugates [2,7,8]. In lieu of widespread diseases and infections, the role of lectins as diagnostic/therapeutic agents in biomedical arena has been of wide interest currently [9–12]. Lectins have been reported amongst varied life forms including plants, animals and microorganisms from fungi, yeasts, bacteria, protozoa, etc. [13]. A repertoire of lectins has been reported from fungal sources including mushrooms [14], microfungi [15] and yeasts [16]. Various fungal lectins exhibit many physiological effects and biomedical applications [17]. Microfungal lectins are emerging as valuable anticancer agents and as novel cancer biomarkers [18–20]. Owing to their remarkable anti-tumor property, lectins have received rave attention from cancer biologists. Microfungal lectins are involved during various cell-cell interactions [21], mitogenesis [22,23], antiproliferative, apoptotic [20] and immunomodulation of immune cells [24,25]. They have the potential to regulate the components of the immune system and modulate immune response, however their immunomodulatory influence have been addressed by only a few authors [25–27]. Lectins from varied microfungal sources exhibit structural diversity which in turn influences their carbohydrate binding specificities [28]. Thus owing to their varied carbohydrate specificity, microfungal lectins act as potential tools in biotechnology, as well as diagnosis/pharmacological and therapeutic applications. This review aims to highlight the sources, characteristics and structural aspects of microfungal lectins and their biomedical applications. 2. Sources of microfungal lectins and their haemagglutination activity A large number of microfungal lectins have been isolated and characterized over the years [15]. They have attracted considerable attention owing to their biomedical potential, however a plethora of microfungal lectins still remains unexplored. Amongst microfungi, lectins from genera Aspergillus [27,29–33], Cephalosporium [21,34], Fusarium [35–38], Penicillium [22,23,39–42], Rhizoctonia [19,43] and Sclerotium [20,44–46] have been widely studied. Microfungal lectins are mostly located in the mycelia [29,33,38–40], however lectin activity has also been reported in the spores [47]. Lectin activity in the culture filtrate of some microfungi has also been reported [48,49]. High lectin content occurs in the mycelia of Sclerotium rolfsii as compared to culture filtrate [50]. S. rolfsii lectin content was abundant in the cytoplasm and nucleus of the vegetative mycelia in media with low C/N ratios grown for 5–11 days [50]. However, lectin content in Rhizoctonia solani strains is higher in sclerotia as compared to mycelia [51]. Lectin might serve as a storage protein in the resting structures of R. solani as indicated by higher lectin concentration in its sclerotia, and during developmental regulation [52]. The ability of lectins to interact with carbohydrate moieties on red blood cell surface and cause agglutination without altering their properties is termed as haemagglutination. The key element of this interaction is strength and specificity of CRD towards erythrocyte saccharide unit [1]. Lectin activity amongst microfungal species has been determined through haemagglutination assay with native/enzyme-treated erythrocytes [15]. Haemagglutinating activity of intracellular lectins from various Aspergillus [29,33,53], Fusarium [37,38] and Penicillium species [40–42] have been thoroughly explored. Based on haemagglutination assay, lectins can fall either in the category of non-specific or specific lectins. Lectins from Aspergillus niger, A. nidulans [29], A. acristatus,

A. gorakhpurensis [33], Macrophomina phaseolina [49] and Sclerotium rolfsii [54] are mostly panagglutinins (non-specific lectins), which agglutinate normal human erythrocytes (A, B, AB, O) almost equally. These lectins might interact with saccharide units other than blood group determinants present on erythrocyte surface. However, endophytic fungal lectins from A. flavus, F. moniliforme, F. oxysporum, Penicillium sp. Trichothecium sp. are blood group specific [55]. Most microfungal lectins have higher agglutination titre with rabbit erythrocytes as compared to human erythrocytes [15,40]. Lectins from Fusarium acutatum, F. lactis, F. globosum, F. proliferatum, F. nivale, F. robustum and F. pseudoanthophilum have been reported to exhibit haemagglutination activity with rabbit erythrocytes only [38]. Majority of microfungal lectins are sensitive to native erythrocytes, however Fusarium solani [35] and Sclerotium rolfsii [56] lectins agglutinate only enzyme treated erythrocytes. 3. Characteristics of microfungal lectins Microfungal lectins have great diversity in their characteristics. Molecular weight of microfungal lectins ranges from 6.5 kDa [57] to 129 kDa [58] with mostly between 25 and 70 kDa [22,23,32,35,39,59–62]. Microfungal lectins are mostly dimeric, either as homodimeric [35,60,62] or heterodimeric [22,23,39,48]. Few monomeric microfungal lectins are also reported [32,49,57,61]. Exceptionally Rhizoctonia crocorum lectin is a tetramer [63] and Rhizopus stolonifer lectin is a hexamer [64]. Microfungal lectins exhibit broad specificity towards varied carbohydrates and glycoproteins [15]. Sialic acid specific lectins have been reported from Aspergillius terricola [32], A. fumigatus [59], A. panamensis [65], Chrysosporum keratinophilum [47], Penicillium duclauxii [22], P. proteolyticum [23], P. marneffei [66] and Trichophytom rubrum [57]. A few microfungal lectins have specificity towards monosaccharides and their derivatives [27,39,61,67,68]. Cephalosporium curvulum lectin has exclusive specificity towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins [69]. The kinetics of lectin-glycan binding have been reported for few microfungal lectins [70]. Affinity constant (Ka) of microfungal lectins can be determined either through surface plasmon resonance or spectrofluorometry [71,72]. F. solani lectin exhibits high affinity constant (Ka 1.61 e10 M−1) for glycoproteins, whereas very low value is shown for simple sugars [71,72]. High affinity constant (Ka) is exhibited by A. oryzae lectin for α-1,6-fucosylated oligosaccharides [73]. Metal ion requirement is not a general characteristic of microfungal lectins [15,22,23,35,58,62,65], however a few are reported as metal ion dependent [49,61]. No effect of EDTA treatment has been reported on lectin activity of Aspergillus panamensis [65], A. gorakhpurensis [62], A. terricola [32], A. nidulans [74], A. sparsus [75], F. solani [35], P. duclauxii [22] and P. proteolyticum [23]. Microfungal lectins usually retain maximum hemagglutinating activity in pH range of 6–8 [22,23,49,61,62,65], however F. solani lectin [35] exhibit exceptional stability over wide pH range (2−12). Stability at temperature range of 20–30 °C is exhibited by most microfungal lectins [22,23,32,62,65], however a few such as Aspergillus flavus, Alternearia sp., Fusarium oxysporum and Trichothecium sp. lectins are stable upto 50 °C [61]. Exceptional stability upto 70 °C is exhibited by Trichophyton rubrum lectin [57] and upto 90 °C by F. solani lectin [35]. High stability of T. rubrum lectin have been attributed to its high glycan content (18%) which might protect the lectin from heating action [57]. Majority of microfungal lectins are glycoproteins, with carbohydrate moiety playing an essential role in their structure and activity. Carbohydrate content varies amongst microfungal lectins amounting to 18% from Trichophyton rubrum [57], 10.4% Macrophomina phaseolina [49], 9% Aspergillus gorakhpurensis [62], 4.95% A. panamensis [65], 3.95% Penicillium duclauxii [22] and 2.83% P. proteolyticum [23], however lectins from Rhizopus stolonifer and Sclerotinia sclerotiorum lack carbohydrate domain [64,76].

R.S. Singh et al. / International Journal of Biological Macromolecules 134 (2019) 1097–1107

4. Structural aspects of microfungal lectins Three-dimensional structures of various lectins have been reported in the Protein Data Bank (PDB, http://www.rcsb.org/pdb/). Structural aspects of classification of CRD of lectins into families have been given in the protein family database, (Pfam, http://pfam.xfam.org/family/). Structural variations amongst microfungal lectins determined through diffraction studies and sequence similarities are depicted in Table 1. They usually exhibit homodimeric configuration with varied number of carbohydrate binding sites and organization. Their amino acid sequence length can range from 141 [68] to 314 [77]. However, a limited reports pertaining to structural details of microfungal lectins are available till date. A thorough understanding of structural organization of lectins and their carbohydrate interaction is necessary as it influences their biological activity. 4.1. Three dimensional (3D) structure of microfungal lectins Three dimensional structural analysis of lectins is determined by Xray crystallography which is the most powerful technique. Various families such as galectins, C-type lectins, leguminous lectins, L-type lectins, P-type lectin families include plant and animal lectins [1]. Fungal lectins analyzed till date have been categorized into having six bladed βpropeller fold, β-trefoil fold or actinoporin like fold [78]. 3-D structures of microfungal lectins along with their family categorization are shown in Fig. 1. Structurally diverse microfungal lectins have been classified into families based on their sequence identities [79]. 4.1.1. Fungal fucose-specific lectin family This family involves lectins which recognize specifically fucosylated glycans and are referred in the PFAM database as Fungal_lectin, PF07938 (http://pfam.xfam.org/family/PF07938). These are also referred to as fungal fucose specific AAL-like family [79]. Hololectin AAL is a dimeric fungal protein from mushroom Aleuria aurantia in which each monomer is organized into six-bladed β-propeller fold. These folds are involved in fucose recognition and consist of five binding pockets between its blades. It also consists of small antiparallel βsheet which is involved in protein dimerization [80]. Lectins from microfungi such as Aspergillus fumigatus, AFL [77] and A. oryzae, AOL [81] belong to this family and form the six-bladed β-propeller organization. These are characterized mainly by the β-strands forming highly repetitive, wheel like structures. AFL adopts a six-bladed β-propeller fold forming a dimeric arrangement with each monomer consisting of six active binding sites [77]. Although, the fold and primary sequence of AAL and AFL are almost similar, however; there is slight variation in the amino acid composition of AFL's binding sites. This difference leads to

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striking variation in its ligand recognition. AFL possess six binding sites which are functionally non-equivalent, whereas AAL possess five binding sites [77]. AOL also consists of six fucose binding sites with each site having varied affinity for fucose as determined with βseleno-fucoside by SAD method [81]. Each site is located between two blades, wherein one blade provides hydrophobic interactions and other contributes to hydrophilic interactions with fucose moiety [81]. To determine the phase of each reflection, multi-wavelength anomalous dispersion (MAD) and single-wavelength anomalous dispersion (SAD) along with selenium incorporated fucoses have been used for structural analysis of AOL-carbohydrate complex [81,82]. Both AOL [81] and AFL [77] have homodimer configuration with 12 carbohydrate binding sites per oligomer. They have 90% similarity towards each other and are thus highly homologous.

4.1.2. Ricin B-chain like family (R-type) These include protein families with structure and sequence similarity to B-subunit of ricin [83]. Ricin is a highly potent toxic protein from Ricinus communis and also known as β-trefoil-type lectin. These are referred in the PFAM database as RicinB_lectin_2, PF14200 (http://pfam. xfam.org/family/PF14200). β-trefoil fold harbors 3 canonical carbohydrate binding sites along with non-canonical sites [84]. Around 150 amino acid residues compose β-trefoil-type lectin [78]. Fungal lectins related to ricin B chain include Clitocybe nebularis, CNL [85], Rhizoctonia solani agglutinin, RSA [86] and Sclerotinia sclerotiorum agglutinin, SSA [87]. These come under β-trefoil type lectins as they have structural and sequence similarity to lectin domain of ricin B. In case of CNL, only one canonical carbohydrate binding site is functional. RSA adopts a βtrefoil fold (not stabilized by disulfide bond) in which each molecule consists of three subdomains (α, β and γ) and assembles around a pseudo 3-fold axis in 3-lobed organization forming a six-stranded βbarrel [86]. Each RSA molecule forms an up and down structural motif comprising of antiparallel β-strands connected by large loops along with 4 small helical regions [86]. SSA also adopts a ß-trefoil domain (not stabilized by disulfide bond) with 3 four-stranded β-sheets (subdomains α, β, and γ) displaying characteristic pseudo-3-fold symmetry, however the hydrophobic character and shape of its carbohydrate binding site are distinctly modified compared to rest of the β-trefoil fold family members [87]. Contrasting to ricin B-type lectin family members, SSA exhibits a novel dimeric assembly and might be involved in multivalent lectin-carbohydrate cross-linking interactions [87]. β-trefoil fold usually harbors three canonical carbohydrate binding sites [83], however RSA contains two functional sites [86] and SSA contains single carbohydrate binding site at α repeat [87].

Table 1 Structural analysis of microfungal lectins. Lectin family

Lectin source

Pfam family

PDB code

Resolution Sequence length

Chain (s)

Homo 2-mer-A2 A,B,C C2221 Homo 2-mer-A2 A P6122 Homo 2-mer-A2 A,B C121 Homo 2-mer-A2 Homo A,B,C,D P61 2-mer-A2 A,B P42212 Homo 2-mer-A2 A,B P42212 Homo 2-mer-A2 A,B P42212 Homo 2-mer-A2

Fungal fucose-specific Aspergillus lectin family fumigatus Aspergillus oryzae A. oryzae

PF07938 4UOU

2.4 Å

314

5EO7

2.3 Å

310

5EO8

1.6 Å

311

Ricin B-chain like family (R-type)

PF14200 4G9M 1.6 Å

143

Fungal fruit body (ABL-like) family

Rhizoctonia solani Sclerotinia sclerotiorum Sclerotium rolfsii S. rolfsii variant 1 S. rolfsii variant 2

2X2S

1.6 Å

153

1.11 Å

142

4YLD

1.7 Å

141

4Z2F

1.6 Å

142

PF07367 2OFC

Space group

A,B,C,D P21

Form

Structural features

Reference

Six-bladed β-propeller

[77]

Six-bladed β-propeller

[81]

Six-bladed β-propeller

[81]

β-trefoil fold

[86]

β-trefoil fold

[87]

2 β-sheets, consisting of 4 and 6β strands, connected by 2 α-helices Antiparallel packed, β-sheets inclined ~45°, α-helices on same side of dimer Antiparallel packed, β-sheets inclined ~45°, α-helices on same side of dimer

[89] [68] [68]

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(a)

(b)

(A)

(c)

(B)

(C)

(d)

(e)

(f)

Fig. 1. 3D Structures of microfungal lectins: (A) Fungal fucose specific family (PF07938): (a) Aspergillus fumigatus lectin (PDB code 4UOU), (b) A. oryzae lectin (PDB code 5EO7), (c) A. oryzae lectin (PDB code 5EO8), (B) Ricin B-chain like (R-type) family (PF14300): (d) Rhizoctonia solani lectin (PDB code 4G9M), (e) Sclerotinia sclerotiorum lectin (PDB code 2X2S) and (C) Fungal fruit body lectin family (PF07367): (f) Sclerotium rolfsii lectin (PDB code 2OFC). Source: RCSB Protein Data Bank (www.rcsb.org) and Pfam (http://pfam.xfam.org/family/).

4.1.3. Fungal fruit body (ABL-like) family Fungal fruit body (ABL-like) family referred to FB_lectin, PF07367 (http://pfam.xfam.org/family/PF07367) in the PFAM database consist of various fungal fruit body lectin proteins. Agaricus bisporus lectin (ABL) is a homotetramer protein in which each monomer is structured into an α/β-sandwich arrangement. It consists of a novel fold having two β-sheets which are joined by a helix-loop-helix motif [88]. This family exhibits structural homology towards actinoporins (family of pore forming proteins from sea anemones). Representatives of this family include Sclerotium rolfsii lectin (SRL), in which six and four β-strands form two β-sheets connected by a helix-loop-helix motif [89]. SRL has two carbohydrate binding sites, wherein primary site recognizes GalNAc and secondary site have specificity towards GlcNAc [89].

4.1.3.1. Molecular and structural biology of Sclerotium rolfsii lectin variants. SRL has low solubility and upon storage, its physio-chemical properties get altered leading to aggregation and loss of activity. In an intention to overcome these problems, full length synthetic genes which encode variant forms of SRL (SSR1 and SSR2) were chemically constructed [68]. In case of SSR1, amino acids Asn, Glu and Glu at 14th, 113th and 123rd positions in SRL were replaced by Asp, Gln and Gln, respectively in SSR1. Amino acids Val, Asp, Ser, Gln and Gln replaces SRL amino acids at the 1st, 14th, 34th, 113th and 123rd positions, respectively in case of SSR2. Thus changes in amino acid sequence were introduced to alter its physio-chemical properties pertaining to enhancing its solubility by altering the surface charge. Further, owing to significance of SRL in recognizing cancer-associated TF antigen, carbohydrate binding

R.S. Singh et al. / International Journal of Biological Macromolecules 134 (2019) 1097–1107

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Microfungal lectins can act as therapeutic and diagnostic agents as they exert varied physiological responses such as antiproliferative, apoptotic, mitogenic, immunomodulatory and cytotoxic effects against cancer cells [15]. They are also endowed with diverse antimicrobial, antioxidant activities, etc. which are discussed ahead:

death and arrests cell in G2/M transition [94]. Thus, SRL treatment shifts cancer cells to hypodiploid/apoptotic phase in time dependent manner as depicted by increased hypodiploid population and reduced cell population in all phases (G0/G1, S and G2/M) of cell cycle [20]. Rhizoctonia bataticola lectin also exhibits growth inhibitory and apoptotic effect on human ovarian [43], human leukemic [95] and colon epithelial cancer cells [19]. RBL exhibits strong binding towards primary and metastatic colon cancer cells and also inhibits their clonogenicity, however it exhibits no binding towards normal colon cells [19]. RBL arrests colon cancer cell growth in S phase, generates ROS and induces apoptosis in colon epithelial cancer cells through activation of caspase-3 [19]. Apart from apoptotic effect, RBL also inhibits metastasis and angiogenesis in ovo [19]. Thus, RBL can be developed as multi targeting anticancer molecule and has potential as diagnostic tool to achieve desired therapeutic effect [19]. A valuable tool for cancer treatment thus involves apoptosis (cell death) in cancer cells involving various molecular and morphological variations. Thus, lectins with potential to distinguish cancer associated antigens and induce apoptosis seem to be promising cancer therapeutic agents.

5.1. Cytotoxic activity of microfungal lectins on cancerous cells

5.2. Effect of microfungal lectins on proliferation of immune cells

Lectins can recognize altered glycans (correlating with malignant transformation) on cancer cell surface and inhibit cell proliferation [17]. Cytotoxic effects of lectins from Ascomycetes on various cancer cell lines are tabulated in Table 2. There has been increased interest in role of lectins as anti-tumor agents having specificity towards cancer cell surface glycoconjugates. Cytotoxic effect of microfungal lectins towards cancerous cells involves generation of signalling cascades upon lectin-glycan interaction leading to disruption of cell cycle and induction of apoptosis [18,20]. Lectin from Aspergillus flavus of Viscum album exhibits caspase-7 mediated apoptosis in human breast adenocarcinoma cell line (MCF-7) through extrinsic pathway [90]. Caspase cleaves the cellular substrates and is the main effector molecule for apoptosis in inactive cells [90]. Sclerotium rolfsii lectin also induces cell apoptosis (in vitro) and suppresses tumor growth (in vivo) upon binding to human colon [44], ovarian [91] and breast cancer cells [45]. As compared to cancerous cells, SRL had minimal effect on normal human epithelial cells [45]. Molecular mechanism pertaining to anticancer effect of S. rolfsii lectin on human colon cells (HT29) involves varied gene expression associated with multiple signalling pathways [18]. Various signalling molecules related with DNA replication, cell cycle, apoptosis, MAPK and c-Jun related show altered expression [18]. The effect of SRL on growth and cell cycle progression in colon cancer cells (HT29 and SW620) showed increased hypodiploid population, reduced cellular cyclin D3 level, reduced cyclin E levels and abolished cyclin D2 and CDK5 expression [20]. Downregulation of cyclin D2 arrests the cells at G0/G1 [92], deregulation of cyclin E is involved in tumorogenesis [93] and inhibition of CDK5 increases cell

Mitogenic lectins can stimulate lymphocyte/splenocyte transformation from resting cells (small) into blast like cells (large). Owing to distinct ability of lectins to interact with cell surface carbohydrates/ glycoproteins, they are known to have proliferative/antiproliferative effects. Several lectins have the ability to show mitogenic activity and induce T helper type 1 (Th1) or type 2 (Th2) responses [7]. The pivotal step in mitogenic induction by lectins involves lectin binding to glycan targets on cell surface. Lectin interaction with T-cell receptor on cell surface commences multitude of reactions involving varied signal transduction pathways involving MAPK or JAK-STAT pathways and ultimately results in cell proliferation [7,96]. Immune response is also induced upon lectin binding, through mediators such as second messengers released from the membrane. Screening of mitogenic capacity of lectins mostly applies splenocytes or lymphocytes model and are evaluated by MTT assay or tritiatedthymidine ([3H]-thymidine) incorporation assay. Amongst microfungi, lectins from Aspergillus, Penicillium, Rhizoctonia and Cephalosporium species are reported to exhibit mitogenic activity (Table 3). R. bataticola lectin exhibits high mitogenic activity towards PBMC at a concentration of 1.25 μg/mL [97], whereas A. nidulans lectin exhibits activity at 200 μg/mL towards splenocytes [74]. However, A. flavus, F. moniliforme, F. oxysporum and Trichothecium sp. endophytic fungal lectins show anti-mitotic depending on the concentration and time of incubation [98]. Considerable reduction in mitotic index values was shown by endophytic fungal lectins at high concentration (100–400 μg/mL) relative to time period, involving alterations in chromosome and cellular morphology. However, low mitotic index value was

specificity of variants was also determined. The two recombinant variants of SRL, SSR1 and SSR2 encode for 141 and 143 amino acids, respectively and correspond to gene lengths of 423 and 426 bps, respectively as demonstrated by restriction digestion analysis [68]. The crystallographic asymmetric unit of both variants of Sclerotium rolfsii lectin consists of two protein molecules namely mol A and mol B packed in antiparallel manner similar to SRL [68]. They are linked by a noncrystallographic two-fold symmetry and the α-helices are located at the same side of the dimer, while β-sheets are inclined ~45° [68]. These two variants only differ in their glycan specificity, where SSR1 exhibits specificity towards TF-antigen and Tn antigen, whereas SSR2 binds only with TF- antigen [68]. 5. Biomedical applications of microfungal lectins

Table 2 Antiproliferative and cytotoxic effect of microfungal lectins. Lectin source

Cancer type

Cell line

IC50 (μg/mL)

Reference

Aspergillus flavus Rhizoctonia bataticola R. bataticola

Human breast adenocarcinoma cancer Human ovarian cancer Human colon cancer

Human ovarian cancer Human leukemic T-cell

Sclerotium rolfsii

Human breast cancer

S. rolfsii S. rolfsii

Human ovarian cancer Human colon cancer

0.02 6.25 5.0 6.4 6.8 5.0 5.0 5.0 20 15 20 10

[90] [97] [19]

R. bataticola R. bataticola

MCF-7 PA-1 HT 29 SW480 SW620 PA-1 Molt-4 Jurkat

IC: inhibitory concentration.

MCF-7 ZR-75 PA-1 DLD-1

[43] [95] [45] [91] [44]

1102

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Table 3 Mitogenic potential of microfungal lectins. Lectin source

Cell type

Mitogenic assay

Lectin concentrationa (μg/mL)

Reference

Aspergillus gorakhpurensis A. nidulans A. panamensis A. sparsus Cephalosporium sp. Penicillium duclauxii P. proteolyticum Rhizoctonia bataticola

Mice splenocytes Mice splenocytes Mice splenocytes Mice splenocytes PBMC Mice splenocytes Mice splenocytes PBMC

MTT assay MTT assay MTT assay MTT assay [3H]-thymidine incorporation assay MTT assay MTT assay [3H]-thymidine incorporation assay

150 200 100 100 10 75 50 1.25

[62] [74] [65] [75] [34] [22] [23] [97]

PBMC: Peripheral blood mononuclear cells; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide. a Lectin concentration showing highest proliferation.

shown at lower lectin concentration (50 μg/mL) and was thus not much effective [98]. Thus, mitogenic lectins have drawn greater attention owing to their ability to exhibit interesting physiological effect involving stimulation of spleen cells including lymphocytes. Further, owing to lectin-lymphocyte interactions, mitogenic lectins can be used for evaluation of immunomodulatory action as T and B lymphocytes are associated to innate and humoral immunity, respectively.

5.3. Immune stimulating effect of microfungal lectins Immune system is the foremost barrier during disease prevention and is involved in protection of an organism against an invasive agent. Innate and adaptive immune responses act by inactivating the aggressive agent [99]. Activation of immune cells, inflammation, and humoral response is modulated by group of cells (neutrophils, eosinophils, basophils, and monocytes/macrophages) and their ability to produce cytokines [99]. Immunomodulators are currently of much interest for improvement of immune conditions in diverse immune responserelated diseases. Some microfungal lectins have the ability to modulate (up or down) immune response mechanism during such immune related conditions. Lectin-glycan interaction can trigger various biochemical pathways and modulate the cascade of proinflammatory and regulatory cytokines. RBL stimulates the proliferation and secretion of Th1/Th2 cytokines (IL2, IFN-γ, IL-4 and IL-10) in human peripheral blood mononuclear cells [100]. The signalling mechanism underlying the immunomodulatory activity of RBL involves p38 MAPK (mitogen activated protein kinase) and STAT-5 (signal transducers and activators of transcription) signalling [100]. RBL can also induce differentiation in human monocytes and monocytic cell line-TPH-1 to macrophages via involvement of NFκβ pathway and thus can be used as in vitro model for macrophage biology study [26]. A. flavus lectin induces interleukin-8 (IL-8) expression which is a proinflammatory cytokine, in a dose dependent manner [27]. IL-8 is associated with p38 MAPK which in turn regulates the expression of activator protein-1 (AP-1) and affect cell expression [27]. AOL has the ability to bind the fucose residue of mast cell bound immunoglobulin (Ig) E [101]. This binding causes antigen independent IgE mediated anaphylactoid reaction (in vivo) and mast cell activation (in vitro), thus suggesting its implication in allergic response to A. oryzae in humans [101]. A. panamemsis [25] and A. gorakhpurensis [24] lectins inhibit systemic anaphylaxis and Arthus reaction, enhances functional ability of macrophages and stimulates cytokine production such as IFN-γ and IL-6 in Swiss albino mice. Sytemic anaphylaxis is an immediate hypersensitivity reaction and Arthus reaction is a manifestation of type III hypersensitivity reaction upon exposure to an antigen [99]. IFN-γ plays role in inflammation and activates macrophages which are important members of innate immunity. Th-2 cytokine IL-2 has both anti-inflammatory and pro-inflammatory activities [99]. As all of these parameters are indicative of immunomodulatory activity, hence Aspergillus and Rhizoctonia lectins have immune stimulating potential. Thus, role of various signalling pathways such as MAPK/STAT,

involved in immunostimulatory potential of lectins could be exploited to modulate immune response during host-pathogen interactions. 5.4. Therapeutic potential of microfungal lectins There has been an increasing interest in pursuit of immune stimulating lectins as therapeutic agents. Amongst microfungi, Aspergillus lectins have shown in vivo therapeutic effect on immune mediated disease, ulcerative colitis [25,102]. Ulcerative colitis is a destructive, chronic inflammatory bowel disease having highest prevalence in world population [103]. It is caused in genetically susceptible host due to abnormal mucosal immune response to intestinal microbiota [103]. A. nidulans [102] and A. panamensis [25] lectins showed curative effect on TNBS acid induced ulcerative colitis in male Wistar rats, thus ameliorating immune status of animals. Intraperitoneal administration of lectins exhibited therapeutic effect against acute mucosal injury. Thus, use of lectins as therapeutic agents (pre-treatment and post treatment) resulted in the increased survival of rats, under disease induced condition. A. nidulans [102] and A. panamensis [25] lectins exhibit high specificity towards N-acetylglucosamine (GalNAc) and L-fucose residues, respectively. High expression of GalNAc in ulcerative colitis gland cells and L-fucose in normal gland cells and inflammatory cells in ulcerative colitis was presumed to the site of lectin interaction. This interaction might have exerted curative effect on animals with experimental colitis via immunomodulation [25,102]. Thus, the immunomodulatory microfungal lectins can be exploited to improve the host immune response against various infections. Considering the therapeutic potential of microfungal lectins, their practical utility in clinical setting would however require further detailed studies pertaining to development of delivery system to improve efficacy and lower toxicity along with immunogenicity studies. 5.5. Antimicrobial activity of microfungal lectins Lectins interact with glycans present over microbial cell surface. The varied glycoconjugates including glycolipids, glycoproteins and polysaccharides possess structural similarity to lectin specific carbohydrate and is thus potential lectin reactive site. Lectins of varied carbohydrate specificity have the ability to inhibit microbial growth or induce death. Although a large number of microfungal lectins have been reported till date, only a few have been analyzed for their antimicrobial activity (Table 4). 5.5.1. Antibacterial activity Gram positive bacteria consist of teichoic acid and peptidoglycans on cell surface. Gram negative bacteria have lipopolysaccharides and peptidoglycans in periplasmic layer [104]. Lipopolysaccharides are composed of a lipid, an O-antigen polysaccharide chain and oligosaccharide core. Peptidoglycans are formed by β(1 → 4) linked MurNAc and Nacetylglucosamine alternated residues, with MurNAc associated tetrapeptides such as L-alanine, D-glutamic acid, meso-diaminopimelic

R.S. Singh et al. / International Journal of Biological Macromolecules 134 (2019) 1097–1107

1103

Table 4 Antimicrobial activity of microfungal Lectins and their carbohydrate specificity. Lectin source

Glycan specificity

Target Inhibition microorganism/s zone

Reference

Diameter

(mm) AmphotericinB –Sclerotium rolfsii Lectin complex Aspergillus flavus

N.S. D-Galactose

and N-Acetyl-galactosamine

Candida albicans Candida glabrata Klebsiella pneumonia Proteus mirabilis Serratia marcescens Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Saccharomyces cerevisiae Bacillus cereus Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Aspergillus niger

A. gorakhpurensis

D-Ribose, L-Rhamnose, D-Glucose, D-Fructose, D-Mannitol, L-Arabinose, D-Sucrose, Chondroitin-6-sulphate, D-Trehalose dehydrate, D-Glucosamine HCl, N-Acetyl neuraminic acid, N-Glycolyl neuraminic acid, N-Acetyl-D-galactosamine, 2-Deoxy-D-Ribose, Fetuin, PSM, Pullulan, Starch, Dextran

A. panamensis

D-Ribose, L-Rhamnose, D-Raffinose, L-Fucose, D-Arabinose, D-Fructose, D-Mannose, D-Sucrose, Inositol, Meso- inositol, D-Galacturonic acid, N-Acetyl neuraminic acid, N-Glycolyl neuraminic acid, 2-Deoxy-D-Ribose, Fetuin, PSM, γ-Globulin

Fusarium acuminatum

L-Fucose, D-Galactose, D-Glucose, D-Glucuronic acid, D-Galacturonic acid, BSM, Thiodigalactoside, Melibiose, Pullulan, Dextran, γ-Globulin

F. chlamydosporium

D-Ribose, L-Rhamnose, L-Fucose, L-Arabinose, D-Galactose, D-Fructose, D-Glucose, D-Glucuronic acid, 2-Deoxy-D-ribose, PSM, Melibiose, Pullulan, Dextran, Inulin, Starch, γ-Globulin

Bacillus cereus Aspergillus niger Candida albicans

F. coeruleum

L-Fucose, D-Galactose, D-Fructose, D-Trehalose dehydrate, Inositol, Meso-Inositol, D-Glucosamine hydrochloride, D-Galactosamine hydrochloride, BSM, PSM, Thiodigalactoside, Pullulan, Dextran,

Bacillus cereus Escherichia coli Staphylococcus aureus Aspergillus niger

F. compactum

D-Fructose, D-Mannitol,

F. crookwellense

L-Fucose, D-Arabinose, D-Galactose, D-Trehalose Dihydrate,

2-Deoxy-D-ribose, BSM, PSM, Fetuin, Asialofetuin, Dextran, Inulin, γ-Globulin

Aspergillus niger

Bacillus cereus Inositol, D-Glucosamine hydrochloride, hydrochloride, Chondroitin-6-sulphate, BSM, Fetuin, Asialofetuin, Thiodigalactoside, Escherichia coli Staphylococcus Pullulan, Dextran aureus Aspergillus niger Candida albicans

D-Galactosamine

F. culmorum

L-Fucose, D-Arabinose, D-Galactose, D-Glucose, D-Galacturonic acid, Thiodigalactoside, Dextran, PSM, N-glycolyl neuraminic acid, Melibiose

Bacillus cereus Escherichia coli Staphylococcus aureus Aspergillus niger Candida albicans

F. Decemcellulare

D-Fructose, D-Mannitol, D-Trehalose Dihydrate, D-Glucosamine hydrochloride, D-Galactosamine hydrochloride, D-Glucuronic acid, D-Galacturonic acid, BSM, N-glycolyl neuraminic acid, Melibiose

Bacillus cereus Escherichia coli Aspergillus niger

F. Dimerium

L-Fucose, D-Arabinose, L-Arabinose, D-Trehalose Dihydrate, D-Galactosamine

F. moniliforme

D-Galactose,

Bacillus cereus Staphylococcus aureus Klebsiella pneumonia Proteus mirabilis Serratia marcescens Staphylococcus aureus

N-Acetyl-galactosamine

hydrochloride, BSM,

N.E. N.E. 13 ± 0.1 9 ± 0.1 9 ± 0.1 12 ± 0.2

[110] [106]

20 ± 0.25 10 ± 0.05 18 ± 0.12 9 ± 0.25

[62]

25 ± 0.25 18 ± 0.05 15 ± 0.75 21 ± 0.12

[65]

18.6 ± 0.57 20 ± 0 13.3 ± 1.15 24 ± 0 20.6 ± 0.57 20.3 ± 1.57 19 ± 1 12.3 ± 0.57 20.3 ± 0.57 19 ± 01 15 ± 1 15.6 ± 0.57 18.6 ± 0.57 19.3 ± 0.57 19 ± 0 16 ± 1 14 ± 0.57 19.6 ± 1.52 10.3 ± 1.52 19.3 ± 1.15 27.3 ± 1.15 20 ± 1 19 ± 1 17.3 ± 0.57 20.6 ± 0.57 19 ± 0.57 14.3 ± 0.57 12 ± 0.2 10 ± 0.1 15 ± 0.2 17 ± 0.1

[37]

[37]

[37]

[37] [37]

[37]

[37]

[37]

[106]

(continued on next page)

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Table 4 (continued) Lectin source

Glycan specificity

Target Inhibition microorganism/s zone

Reference

Diameter

(mm) F. oxysporum

Penicillium corylophilum

P. expansum

P. purpurogenum

Tricothecium sp.

Klebsiella pneumonia Proteus mirabilis Serratia marcescens Staphylococcus aureus Bacillus cereus D-Ribose, D-Raffinose, D-Galactose, D-Glucose, D-Maltose, D-Sucrose, Inositol, Meso-Inositol, Escherichia coli Chondroitin-6-sulphate, BSM, PSM, Asialofetuin Pseudomonas aeruginosa Staphylococcus aureus Aspergillus niger Saccharomyces cerevisiae Bacillus cereus D-Ribose, D-Xylose, D-Fructose, D-Glucose, D-Mannitol, D-Sucrose, D-Galactosamine hydrochloride, Escherichia coli D-Glucuronic acid, N-Acetyl glucosamine, N-Acetyl galactosamine, Chondroitin-6-sulphate, BSM, PSM, Pseudomonas Asialofetuin, Thiodigalactoside, Melibiose, γ-Globulin aeruginosa Aspergillus niger Candida albicans Escherichia coli D-Raffinose, L-Fucose, D-Mannose, D-Glucose, D-Sucrose, D-Trehalose dehydrate, D-Glucosamine Pseudomonas hydrochloride, D-Galactosamine hydrochloride, Chondroitin-6-sulphate, BSM, PSM, Asialofetuin, aeruginosa Melibiose, Starch, γ-Globulin Aspergillus niger Saccharomyces cerevisiae Staphylococcus D-Galactose, N-Acetyl-galactosamine aureus Proteus mirabilis Serratia marcescens Klebsiella pneumonia D-Galactose,

N-Acetyl-galactosamine

14 ± 0.1 11 ± 0.1 13 ± 0.1 18 ± 0.1

[106]

N.S. N.S. N.S. N.S. N.S. N.S.

[42]

N.S. N.S. N.S. N.S. N.S.

[42]

N.S. N.S. N.S. N.S.

[42]

12 ± 0.2 10 ± 0.1 13 ± 0.1 13 ± 0.1

[106]

N.E.: inhibition zone not evaluated (minimum inhibitory concentration: 0.4 μg/mL). N.S.: not specified.

acid, and D-alanine forming cross-links between the peptidoglycan strands [105]. Amongst microfungi, Penicillium corylophilum and P. expansum lectins inhibit both Gram positive and Gram negative bacteria, whereas P. purpurogenum inhibits only Gram negative bacteria [42]. Aspergillus lectins have more pronounced inhibition over Gram positive bacteria as compared to Gram negative [62,65]. Fusarium acuminatum, F. coeruleum, F. crookwellense and F. culmorum lectins also exhibit antibacterial activity against E. coli, B. cereus and S. aureus [37]. Endophytic fungal lectin from F. moniliforme and F. oxysporum exhibits broad antibacterial effect and high growth inhibition towards pathogenic bacteria Proteus mirabilis, Serratia marcescens, Klebsiella pneumoniae and Staphylococcus aureus [106]. F. moniliforme, F. oxysporum and A. flavus endophytic fungal lectins have non-selective activity against tested bacterial pathogens and showed significant inhibition at minimum concentration [106]. Antibacterial activity of lectins in case of Gram negative bacteria might occur due to direct interaction between lectin and bacterial membrane receptors such as N-acetylglucosamine, N-acetylmuramic acid or linked tetrapeptides. In case of Gram positive bacteria, lectin might interact with lipopolysaccharides of cell wall [107]. Lectin-glycan interaction might initiate various indirect mechanisms involving reduced nutrient uptake, alterations in cell permeability, activating intracellular responses, etc. which lead to bacterial growth inhibition [108]. 5.5.2. Antifungal activity Fungal cell wall is composed of polysaccharide chitin (repeating Nacetylglucosamine residues) which is a major constituent along with

glucans and glycoproteins [109]. F. chlamydosporium, F. culmorum and F. crookwellense lectins exhibit antifungal activity against Candida albicans [37]. Sclerotium rolfsii-Amphotericin B complex inhibits both C. albicans and C. glabrata with a minimum inhibitory concentration 0.4 μg/mL [110]. Antifungal activity of lectins can be ascribed to their carbohydrate binding specificity. Chitin binding lectins can inhibit fungal growth [111]. Inability of A. gorakhpurensis lectin to bind fungal cell wall corroborates with its non-specificity towards N-acetyl-D-glucosamine [62]. A few lectins can bind to fungal hyphae leading to poor nutrient absorption and interfere in spore germination thus inhibiting fungal growth [112]. Some lectins can render fungi more vulnerable to varied stress conditions owing to induced morphological variations [113]. Lectin binding to fungal surface carbohydrates can produce some indirect effects such as impairment of chitin synthesis/deposition in cell wall which interrupts cell wall synthesis and impedes fungal growth [109]. 5.6. Antioxidant activity of microfungal lectins Free radicals such as superoxide, hydroxyl, peroxyl, etc. are generated during normal or pathological cell metabolism and have one or more unpaired electrons. In living cells, oxidative stress condition arises primarily due to reactive oxygen species (ROS) derived from oxygen radical and results in cellular damage [114]. Antioxidants are compounds that scavenge free radicals by donating electrons/hydrogen to radicals and thus play significant role in ameliorating diseases related to free radical attack [115]. Various in vitro assays such as 1,1diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging, hydrogen

R.S. Singh et al. / International Journal of Biological Macromolecules 134 (2019) 1097–1107

peroxide scavenging activity and ferric reducing antioxidant power (FRAP) can be used to determine the antioxidant capacity of lectins [55]. Lectins isolated from different endophytic fungi from Viscum album such as Aspergillus flavus, Fusarium oxysporum, F. moniliforme and Trichothecium sp. have antioxidant activity [55]. Antioxidant screening by DPPH assay is one of the basic reproducible methods involving antioxidant reaction with DPPH and its conversion into 1,1diphenyl-2-picryl hydrazine [116]. The percentage inhibition of DDPH scavenging activity shown by endophytic fungal lectin from Aspergillus flavus, Trichothecium sp., Fusarium oxysporum and Fusarium moniliforme was 88.4%, 88.5%, 86.64% and 82.9%, respectively [55]. Antioxidant potential is determined by concentration required to achieve half maximal inhibition represented by IC50. Lower IC50 values depicting greater antioxidant activity were shown by Aspergillus flavus (IC50: 127.9) and Fusarium oxysporum (IC50: 129.3) lectins, as compared to F. moniliforme (IC50: 143.6) lectin [55]. The hydroxyl group present in the endophytic fungal lectin may have inhibited DPPH radical [55]. Endophytic fungal lectin shown hydrogen peroxide scavenging in a concentration dependent manner with lowest IC50 value shown by Fusarium moniliforme [55]. Hydrogen peroxide scavenging by endophytic fungal lectin might be due to the electron donating to H2O2, thus counterbalancing it to water [117]. The FRAP assay is another significant indicator of potential antioxidant activity and mainly depends on the reducing capacity of Fe3+ to Fe2+ [118]. Endophytic fungal lectins from A. flavus, F. oxysporum, F. moniliforme and Trichothecium sp. showed concentration dependent increase in the reducing ability during FRAP assay with lowest IC50 value of 13.6 shown by F. moniliforme [55]. Thus, endophytic fungal lectins can act as potential source to succumb the free radicals and thus have great pharmaceutical value. 6. Conclusions Lectin-carbohydrate interactions play role in many biological processes and act as tools to interpret various cell surface changes. An understanding of haemagglutination activity of microfungal lectins and their physio-chemical characteristics involving stability studies help to explore their potential applications. Further lectin involvement in various applications in based upon their glycan specificity. Studies of lectin structure and lectin-carbohydrate structural interaction shows their relation with lectins from other genera and also help in classification of lectins. Owing to glycan specificity they can either interact with glycans and induce mitogenicity or recognize altered glycans and inhibit cell proliferation in case of cancerous cells. Microfungal lectins have the ability to induce proliferation of immune cells and exert immunomodulatory effect. Owing to their antimicrobial and antioxidant potential, microfungal lectins can be of great interest in pharmaceutical research. They are emerging as potential antimicrobial candidates for drug therapies, although clinical trials pertaining to dosage, delivery and bioavailability are need of the hour. Declaration of Competing Interest The authors report no declarations of interest.

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[18]

[19]

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[21]

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