Protein J (2011) 30:374–383 DOI 10.1007/s10930-011-9342-0
Purification and Characterization of a Lectin with High Hemagglutination Property Isolated from Allium altaicum Santosh Kumar Upadhyay • Sharad Saurabh • Rahul Singh Preeti Rai • Neeraj Kumar Dubey • K. Chandrashekar • Kuldeep Singh Negi • Rakesh Tuli • P. K. Singh
•
Published online: 6 July 2011 Ó Springer Science+Business Media, LLC 2011
Abstract A lectin was purified from the leaves of Allium altaicum and corresponding gene was cloned. The lectin namely Allium altaicum agglutinin (AAA) was *24 kDa homodimeric protein and similar to a typical garlic leaf lectin. It was synthesized as 177 amino acid residues preproprotein, which consisted of 28 and 43 amino acid long N and C-terminal signal peptides, respectively. The plant expressed this protein more in scapes and flowers in comparison to the bulbs and leaves. Hemagglutination activity (with rabbit erythrocytes) was 1,428 fold higher as compared to Allium sativum leaf agglutinin (ASAL) although, the insecticidal activity against cotton aphid (Aphis gossypii) was relatively low. Glycan array revealed that AAA had higher affinity towards GlcAb1-3Galb as compared to ASAL. Homology analysis showed 57–94% similarity with other Allium lectins. The mature protein was expressed in E. coli as a fusion with SUMO peptide in soluble and biologically active form. Recombinant protein retained high hemagglutination activity. Santosh Kumar Upadhyay and Sharad Saurabh Contributed equally to the results of this study. S. K. Upadhyay S. Saurabh R. Singh P. Rai N. K. Dubey K. Chandrashekar P. K. Singh (&) Council of Scientific and Industrial Research, National Botanical Research Institute, Rana Pratap Marg, Lucknow, Uttar Pradesh 226001, India e-mail:
[email protected];
[email protected] R. Tuli Department of Biotechnology, National Agri-Food Biotechnology Institute, C-127, Phase VIII, Industrial Area, SAS Nagar, Mohali, Punjab 160071, India K. S. Negi National Bureau of Plant Genetic Resources, Regional Station, Bhowali, Nainital, Uttarakhand 263132, India
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Keywords Agglutinin Allium altaicum Carbohydrate binding Hemagglutination Insecticidal Lectin Abbreviations AAA Allium altaicum agglutinin ASAL Allium sativum leaf agglutinin EDTA Ethylenediamine tetra acetic acid SUMO Small ubiquitin like modifier RFU Relative fluorescence units GNA Galanthus nivalis agglutinin RT-PCR Reverse transcriptase PCR
1 Introduction Plant lectins, also known as ‘‘agglutinins’’, are heterogeneous group of carbohydrate binding proteins, bind to simple sugars and/or complex carbohydrates, reversibly [30]. Lectins are produced by a diversity of organisms including animals [34], plants [35], fungi [14] and bacteria [21]. Lectins have confined the attention of researchers in view of their potentially exploitable activities like antiproliferative [36], immuno-enhancing [17], anti-fungal [16], anti-HIV [19], insecticidal [24] and others. Several lectins have been isolated and characterized from family Alliaceae, Amaryllidaceae [6], Araceae [11], Bromeliaceae, Liliaceae and Orchidaceae etc. and majority of them are oligomeric, either homo-oligomeric or heterooligomeric [31, 32]. Plant lectins have shown varying degree of insecticidal activity in feeding experiments on artificial diets as well as on transgenic plants. Among previously reported lectins, Allium sativum leaf agglutinin (ASAL) is most studied for the insecticidal activity [24, 28, 29].
Purification and Characterization of Allium altaicum Agglutinin
In the present study, we isolated a lectin from Allium altaicum which did not show significant insecticidal activity as compared to ASAL but had remarkably high hemagglutination activity. We purified A. altaicum agglutinin (AAA), studied its carbohydrate binding properties on glycans array, and predicted three dimensional structure and evolutionary relationship with other lectins. We also studied expression pattern of AAA in source plant. The gene was cloned, expressed in E. coli and biological activity of recombinant protein was examined.
2 Materials and Methods 2.1 Isolation of AAA and Estimation of Molecular Size Allium altaicum leaves were collected from the germplasm collection of National Bureau of Plant Genetic Resources, regional centre, Bhowali, Uttarakhand, India. AAA was purified as described [29] and stabilized in PBS (Phosphate Buffered Saline) for further experiments. The protein was also visualized on 15% denaturing polyacrylamide gel. Molecular size of the purified protein was estimated on size exclusion column, PBS was used as mobile phase. Chymotrypsinogen (25 kDa), Ovalbumin (43 kDa) and Bovine Serum Albumin (66 kDa) was taken as molecular size standards. 2.2 Immuno-Blot and MS/MS Analysis Protein was transferred to PVDF membrane and immunostained with anti-ASAL antibody (1:1,000) and anti-rabbit HRP-conjugate antibody (1:20,000). Color was developed with 3-30 -diaminobenzidine tetrahydrochloride (DAB, GeNeiTM, India). The protein was also resolved on 2D gel, spot excised and MS/MS analysis performed as described [28]. 2.3 Hemagglutination and Glycan Array Analysis Hemagglutination assays were carried out in V-bottom microtitre plates in triplicate. 50 lL of AAA (two-fold serially diluted in PBS) and 50 lL of 2% trypsinized rabbit erythrocytes suspension were mixed gently. Microtitre plate was incubated for 1 h at room temperature and agglutination was observed visually. Reciprocal of the highest dilution of lectin showing detectable agglutination was taken as titer of the hemagglutination. ASAL served as positive control. Glycan array version 4.0 was performed by the Consortium of Functional Glycomics as described [4] (https:// www.functionalglycomics.org/static/consortium/resources/ resourcecoreh8.shtml) with 100 lg/mL concentration of lectin. A polyclonal anti-ASAL antibody labeled with Atto
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488 (ReaMetrix, India) was used for the detection of AAA bound to glycans. The data was reported as average relative fluorescence units from four of the six replicates (leaving highest and lowest values) for each glycan represented on the array. 2.4 Temperature/pH Stability and Metal Ion Requirement The stability of the purified AAA towards temperature and pH was studied as described [29]. The lectin (1 mg/mL) was dialyzed against 0.1 M ethylenediamine tetra acetic acid (EDTA) for 48 h. One part of the EDTA-treated lectin was re-dialyzed against PBS, while other against 100 mM CaCl2, MgCl2 and MnCl2 (separately) for 48 h. Hemagglutination titer of the salt treated and untreated lectin was compared. 2.5 Insect Bioassay Against Aphids (Aphis gossypii) Aphid culture was maintained in the glass house on cotton plants. Bioassay was carried out as reported for whiteflies [27] with some modification. Specimen tubes were 25 mm 9 30 mm (diameter 9 height) in size and sealed. Modification was necessary due to low mobility of aphids as compared to whiteflies. Artificial diet ‘Diet A5’ [10] was mixed with different concentrations of lectins (50 lL lectin ? 950 lL diet), filter sterilized through 0.22 lm membrane filter and sandwiched between two layers of sterilized stretched parafilm on the inner side of the caps of the specimen tube. All the steps were carried out aseptically under laminar flow. Bioassays were carried out with first instar nymphs, 25 nymphs were released in each tube. Experiments were performed in triplicates. Diet was changed on alternate days to avoid contamination in diet and/or degradation of lectins. 2.6 DNA and RNA Extraction from A. altaicum, 30 and 50 RACE DNA was prepared by CTAB method [26]. RNA was prepared with Total RNA Isolation Kit (Sigma, USA) from fresh leaf samples. cDNA synthesis was done with SMART technology (RACE Kit, Clontech Laboratories Inc.). 50 and 30 RACE were performed as per manufacturer’s protocol. For 30 RACE, gene specific primer (50 -AAC/TGGGT GCAGGGCC/TGTG/CATGC-30 , designed from the conserved region of the reported Allium lectins) and Universal Primer A mix (provided with kit) were used. Two more gene specific primers (50 -GGACGGCAGTGACATTCTG ATTC-30 and 50 -CTGGTCCAATCACTGAGCCTCC-30 ) were designed for 50 RACE. Finally, terminal primers (forward 50 -ATGGGCAGCACTCCATCTC-30 and reverse
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50 -TCATGCAGCAGCAGCAACC-30 ) were designed for the amplification of complete gene. Complete gene was also amplified from genomic DNA to detect the presence of intron.
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were analyzed by immuno-blotting. Positive fractions were dialyzed against PBS and used for hemagglutination assay.
3 Results 2.7 Sequence Analysis and Molecular Evolution Analysis ORF finder tool (http://www.ncbi.nlm.nih.gov/projects/ gorf/orfig.cgi) was employed for the identification of ORF region from full-length cDNA. Amino acid sequence of AAA was deduced from encoding DNA using ExPASy translate tool. Signal peptide was analyzed on SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). Analysis and comparison of deduced amino acid was performed on BLAST-P and CLUSTALW, respectively. The conserved domains were searched using RPS-BLAST on NCBI. Phylogenetic analysis was done with other lectins of plant origin using CLUSTALW. 2.8 Molecular Modeling of AAA Secondary structure prediction was carried out by SOPMA secondary structure prediction method [13]. Tertiary structure prediction was carried out on I-TASSER server (http:// zhang.bioinform-atics.ku.edu/I-TASSER/output/S26810/) [37]. 2.9 Expression Profiling Semi-quantitative RT-PCR was done to investigate the aaa transcript in various tissues. RNA was extracted from roots, stems, bulbs, leaves, scapes and flowers and RT-PCR was performed. The amplification of aaa was done with forward (50 -ACGAATACAGCACCCCAATCTG-30 ) and reverse primers (50 -TTCTGCTGTTACTGGCCCAGAC30 ), which amplified a fragment of 150 bp. Amplification of actin served as internal control.
3.1 Purification of AAA AAA was purified to about 80% homogeneity following a well established protocol [25]. It was further purified to 95% homogeneity as described [29]. Approximately, 1 mg of protein was obtained from 500 g of leaf tissue (Fig. 1). The molecular size was estimated by size exclusion chromatography (on Superdex 75 column). Purified AAA was eluted at the volume corresponding to *24 kDa. Results of denaturing polyacrylamide gel and size exclusion chromatography indicated that it existed as a homodimer of approximately 12 kDa subunits. 3.2 Hemagglutination, Glycan Array and Insect Bioassay AAA agglutinated rabbit erythrocytes at a minimum concentration of 0.14 ng/mL, which was 1,428 fold higher compared to A. sativum leaf agglutinin (200 ng/mL) and other reported Allium lectins [25, 29]. The lectin did not require divalent cations for hemagglutination activity. AAA showed highest relative affinity towards the GlcAb1-3Galb, significant affinity towards high mannose N-glycan (Mana1-3(Mana1-2Mana1-2Mana1-6) Mana) and complex N-glycans (Galb1-4GlcNAcb1-2Mana13(Mana1-6) Manb1-4GlcNAcb1-4GlcNAcb). It did not bind to monosaccharides. AAA caused relatively low toxicity to cotton aphid as compared to ASAL. The results are shown in Tables 1 and 2.
2.10 Expression and Purification of Recombinant AAA in E. coli The gene encoding mature protein was cloned into two different E. coli expression vectors (pET-28b and pSUMO), transformed into E. coli strain (BL21) RIL and protein expression was carried out as described [29]. Total soluble protein containing recombinant protein was loaded on HiTrap QXL 1 mL column (GE Healthcare, USA) preequilibrated with 20 mM TrisCl (pH 9.0) at a flow rate of 0.1 mL/min. The column was washed with same buffer till absorbance at 274 nm became 0.002 and stabilized. Proteins were eluted with 0.0–0.1 M linear gradient of NaCl with elution volume 35 mL. The fractions containing lectin
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Fig. 1 Purification of AAA. lane M molecular weight marker, lane 1 crude extract of A. altaicum leaf, lane 2 AAA after affinity purification, lane 3 further purified and concentrated on 10 kDa cutoff filter
Purification and Characterization of Allium altaicum Agglutinin
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Table 1 Glycan array analysis of AAA by the consortium of functional glycomics Glycan Number
Structure
RFU
STDEV
194
GlcAb1-3Galb-Sp8
1,444
49
205
Mana1-3(Mana1-2Mana1-2Mana1-6)Mana-Sp9
528
174
346
Galb1-4GlcNAcb1-2Mana1-3(Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12
527
91
359
Gala1-3Galb1-4GlcNAcb1-2Mana1-3(Gala1-3Galb1-4GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb14GlcNAcb-Sp20
254
31
48
Mana1-3(Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp13
197
83
299
GlcAb1-3GlcNAcb-Sp8
171
49
341
Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Mana1-6)Manb1-4GlcNAcb1-4GlcNAc-Sp12
108
30
198
Mana1-2Mana1-2Mana1-3Mana-Sp9
102
58
337
GlcNAca1-4Galb1-4GlcNAcb1-3Galb1-4(Fuca1-3)GlcNAcb1-3Galb1-4(Fuca1-3)GlcNAcb-Sp0
100
22
Values are average of relative fluorescence units (RFU) from four replicates. Data is available on https://www.functionalglycomics. org/static/consortium/resources/resourcecoreh8.shtml Table 2 Insecticidal activity of ASAL and AAA against Aphis gossypii Percent mortality 2 days Mean ± SE
4 days Mean ± SE
4.0 ± 1.2ab
10.7 ± 0.7ab
ASAL (lg/mL) 10
6.0 ± 0.0
b
12.0 ± 1.2bc
12.0 ± 1.2
c
31.3 ± 1.8d
60
19.3 ± 1.8
d
43.3 ± 1.7e
80
27.3 ± 1.3e
55.3 ± 5.3f
10
1.7 ± 0.3a
5.3 ± 0.7a
20
2.0 ± 1.2
a
4.3 ± 0.9
ab
12.7 ± 0.7b
60
6.7 ± 0.3
ab
20.0 ± 2.0c
80
12.0 ± 1.2c
40.0 ± 1.2e
a
5.0 ± 0.6a
20 40
AAA (lg/mL)
40
Control
2.0 ± 0.0
6.7 ± 0.7ab
Percent mortality was calculated by probit analysis using SPSS (version 10) programme. Values are means of three replicates. They were compared using Duncan’s Multiple Range Test (DMRT) at a = 0.05; means in the column carrying same letter are not significantly different
3.3 MS/MS Analysis Peptide mass fingerprinting data matched with mannosebinding lectin of A. cepa (Nominal mass: 12410 Da; Calculated pI value: 7.93, Number of mass values matched: 10, Sequence coverage: 40%). Result of de novo sequencing of six peptides matched with the primary structure of lectin deduced from the cloned gene; five peptides matched with 100% similarity and one with 92.3% (Table 3).
3.4 Stability, pH and Temperature Optima Purified AAA was stable in PBS for 2 years at 4 °C and at least 6 months at room temperature (data not shown). AAA partially restored the hemagglutination property after heat treatment at 100 °C for 35 min. It was found active over a broad pH range (4–11) and optimum activity was observed at pH 7–8 (Fig. 2a, b, c). 3.5 Cloning and Characterization of the cDNA Full-length cDNA, was 710 bp and had 531 bp long open reading frame (NCBI accession no. HM004355), 38 bp 50 UTR and 110 bp 30 UTR with 46% GC content. Theoretical translation yielded 177 amino acid residues long lectin pre-proprotein with a calculated molecular mass of 18.9 kDa. 28 amino acid residues long sequence at the N-terminus was predicted as signal peptide by using SignalP software (Fig. 3), which was in agreement with N-terminal sequences reported in the literature. Removal of signal peptide resulted into a protein of 16.1 kDa; which indicated the presence of additional peptide at the C-terminus. After theoretical removal of 43 amino acid residues long peptide from C-terminus, the calculated molecular weight of the lectin was 11.8 kDa, which was in agreement with the observed molecular weight on denaturing PAGE. No intron was found in the amplified product of genomic DNA (result not shown). 3.6 Homology Analysis and Structure Prediction R29 to Y136 of AAA was found conserved. It belonged to B-lectin (Bulb type lectin, cd00028) family. BLAST-P analysis showed high homology of lectin with homo-oligomeric lectins (single domain) in comparison to heterooligomeric lectins. The members of this family contained a
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Table 3 Result of de novo sequencing of trypsin digested AAA peptides and their % similarity with the sequence deduced from the cloned gene Peptide identified by MS/MS AVWASNSR
Mr (expt)
Mr (calc)
Sequences of the peptide matched from cloned gene
% identities
AVWASNSR
100
889.4406
889.4406
AVWASNSRR
1,045.5417
1,045.5417
AVWASNSRR
100
GRAVWASNSR
1,102.5631
1,102.5632
GRAVWASNSR
100
GNGNYILVLQKDR
1,489.7524
1,488.8049
GNGNYILVLQEDR
NVVIYGSDIWSTGTYR
1,829.8948
1,829.8948
NVVIYGSDIWSTGTYR
100
NVVIYGSDIWSTGTYRK
1,957.9897
1,957.9898
NVVIYGSDIWSTGTYRK
100
consensus sequence motif (QXDXNXVXY). AAA showed highest identity with A. cepa agglutinin as observed in CLUSTALW analysis. Percent similarity with other Allium lectins like A. cepa agglutinin (AAR23522.1), A. porrum agglutinin (AAC37361.1), A. sativum leaf agglutinin (AAW48531.1), A. triquetrum agglutinin (ABA00714.1), A. sativum agglutinin (AAA32643.1) and A. ursinum agglutinin (AAC37358.1) was 94, 83, 80, 67, 66 and 57%, respectively (Fig. 4a). Predicted secondary structure of AAA pre-proprotein consisted of 16.95% alpha helix, 35.03% extended strand, 14.69% beta turn and 33.33% random coil. Alpha helix was present at the N as well as at C-terminus, whereas rest of the structure consisted of extended strand and random coil (Fig. 4b). Amino acid ‘‘QN’’ of carbohydrate-binding motifs was part of random coil, amino acid ‘‘VY’’ of extended strand, and amino acid ‘‘D’’ of beta turn. Tertiary structure prediction of AAA showed that, the fold consisted of ten anti-parallel b-sheets, two helices and several turns and loops. The fold resembled a prism like structure, formed by b-sheets together with loops and turns, in which helices lied on one side. The predicted structure showed high similarity with the A. sativum agglutinin, ASA (pdb 1bwuA and 1kj1A) (Fig. 4c). 3.7 Molecular Evolution Analysis A phylogenetic tree was constructed on the basis of protein sequence of AAA and other plant agglutinins. All the monocot lectins were grouped into three big clusters (Fig. 5). The first cluster consisted of lectins of several taxonomically distinctly related families such as Alliaceae, Amaryllidaceae, Annonaceae, Araceae, Bromeliaceae, Dioscoreaceae, Hycinthaceae and Orchidaceae. Most of the lectins belonged to family Alliaceae. Second and third clusters grouped lectins from family Araceae and Amaryllidaceae, respectively. 3.8 Expression of aaa in Various Tissues Semi-quantitative RT-PCR showed expression of aaa in all the parts of plant except root and stem. Relatively high
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92.3
expression was found in scapes and flowers in comparison to the bulb and leaves (Fig. 6). 3.9 Recombinant Expression of AAA in E. coli The expression of AAA from pET28b vector was poor and protein accumulated in insoluble inclusions (data not shown). The protein was expressed reasonably well with SUMO fusion (at the N-terminus). The protein was soluble and had biological activity (Fig. 7a). Surprisingly, AAA did not bind to Ni–NTA column. Protein was purified partially on HiTrap QXL column. Hemagglutination activity established the presence of recombinant AAA in peak 1 (Fig. 7b). It was also confirmed with immune-blot analysis (Fig. 7c). Minimum agglutination concentration of recombinant AAA was approximately 1 ng/mL as compared to 200 ng/mL of ASAL.
4 Discussion Carbohydrate binding lectins derived from plants are known to offer reasonable control against several insect pests [22, 33]. Among the plant derived lectins, ASAL is the most preferred insecticidal protein for the development of insect resistant transgenic plants. ASAL is toxic against spectrum of insects of order Homeoptera and Lepidoptera [1, 8]. Researchers are still discovering a lectin with better toxicity from edible sources and trying to correlate hemagglutination with insecticidal activity. AAA purified from A. altaicum leaf did not show significant insecticidal activity however, it had very high hemagglutination property (against rabbit erythrocytes) in comparison to ASAL. Other properties like stability, temperature and pH optima were comparable. Like other mannose binding lectins of Allium species, AAA is a homodimer. There are a few exceptions of trimeric and tetrameric forms of lectins also (isolated from A. cepa and A. porrum, respectively) [2]. AAA showed high affinity towards GlcAb1-3Galb and reasonable affinity for high mannose N-glycan (Mana1-3(Mana1-2Mana1-2Mana1-6)Mana) and complex
Purification and Characterization of Allium altaicum Agglutinin
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Fig. 3 Nucleic and amino acid sequences of AAA. Figure shows 28 amino acid long N-terminal signal peptide, 43 amino acid long C-terminal peptide (in red rounded rectangle) and three carbohydrate binding domains (in blue rounded rectangle)
Fig. 2 Heat and pH stability of AAA. a Hemagglutination activity after heat treatment for 10 min at different temperatures, b activity after heat treatment at 100 °C for different time interval, c activity after incubation of AAA at different pH for 24 h
N-glycan (Galb1-4GlcNAcb1-2Mana1-3(Mana1-6)Manb14GlcNAcb1-4GlcNAcb) in contrast to ASAL which showed high affinity towards complex N-glycan (Galb14GlcNAcb1-2Mana1-3(Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb)
and some affinity towards GlcAb1-3Galb and high mannose N-glycans [29]. Although there was significant difference in the glycan affinity profile of these two lectins, Mannose was a common residue in most of the interacting glycans (Table 1; [29]). This was probably the reason of purification of both the lectins on Mannose-Agarose column. Surprisingly, none of the lectins showed affinity towards Mannose in monosaccharide form on glycans array. Spatial availability of Mannose in matrix and array were probably different. There are three carbohydrate binding domains in Allium lectins. Differential affinity and specificity of lectins on glycan array indicated that each domain might have affinity towards one or a few glycans. It is noteworthy that the carbohydrate binding domains in lectins are highly conserved; little variation can alter their carbohydrate binding property. Mannose is abundant sugar in the glycans of gut proteins of the insect [18]. AAA showed highest relative affinity with glycan (GlcAb1-3Galb) which is uncommon in insects and low affinity towards other mannose containing complex glycans. In contrast ASAL interact mostly with the mannose containing glycans [29]. This might be a one of the reasons of low toxicity of AAA to cotton aphids. Hemagglutination property of lectins is a consequence of their interactions with glycans present on membrane of erythrocytes [5]. In our earlier studies, we demonstrated affinity of ASAL and SUMO-ASAL towards complex
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Fig. 4 Homology of AAA with other Allium lectins and its predicted structure. a Multialignment of the deduced amino acid sequence of AAA with six most homologous monocot mannose binding lectins from A. cepa (AAR23522.1), A. porrum (AAC37361.1), A. sativum (AAW48531.1), A. triquetrum (ABA00714.1), A. sativum (AAA32643.1) and A. ursinum agglutinin (AAC37358.1). Dark area represents carbohydrate binding domains. b Secondary structure of AAA. The helices, sheets, turns and coils are indicated with blue, red, green and violet vertical lines, respectively. c Tertiary structure of AAA. a-helices and b-sheets are indicated as red ribbon and yellow arrow symbols, while irregular loops and turns are shown in white and blue lines, respectively
carbohydrates on glycan array platform. They differ in their carbohydrate binding property (among 10 highest affinity glycans, only three glycans, no. 346, 393 and 194 were common). Comparable hemagglutination property of both the proteins indicated good possibility of involvement of 346, 393 and 194 type interaction between ASAL and rabbit erythrocyte [29]. Glycans 346 and 393 consisted of Galb14GlcNAcb1-2Mana1-3 residues which featured presence of N-acetyllactosamine (Galb1-4GlcNAc). N-acetyllactosamine has been reported as major constituent of N-linked carbohydrate chain on surface proteins of mammalian
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erythrocyte [20]. This indicated the role of N-acetyllactosamine in hemagglutination activity. AAA not only interacted with glycans 346 but also with another glycan (359) where galactosyl residue is attached to N-acetyllactosamine through a-1-3 linkage (Table 1). This interaction might have caused higher agglutination. Presence of galactosyl residue at the terminal position in Galb1-4GlcNAc is a characteristic feature of rabbit erythrocytes. It is noteworthy that Glycans of other mammalian erythrocytes are mostly capped with Sialic acid [9, 20]. Affinity towards glycan 359 is an exclusive feature of AAA.
Purification and Characterization of Allium altaicum Agglutinin
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Fig. 5 The phylogeny of lectins from families Alliaceae, Amaryllidaceae, Annonaceae, Araceae, Bromeliaceae, Dioscoreaceae, Hernandiaceae, Hyacinthaceae, Iridaceae, Liliaceae, Orchidaceae and Ruscaceae. The lectin sequences were downloaded from NCBI, and the accession numbers are as follows: ACA from A. cepa, Alliaceae (AAR23522); APA from A. porrum, Alliaceae (AAC37361); ASA from A. sativum, Alliaceae (AAA32643); ASAL from A. sativum, Alliaceae (AAW48531); AUA from A. ursinum, Alliaceae (AAC 37358); ATA from A. triquetrum, Alliaceae (ABA00714); AVA from Amaryllis vittata, Amaryllidaceae (AAP57409); CAA from Crinum asiaticum, Amaryllidaceae (AAO59506); CMA from Clivia miniata, Amaryllidaceae (AAA19910); GNA from Galanthus nivalis, Amaryllidaceae (AAL07474), HTA from Hippeastrum sp. Amaryllidaceae (AAA33362); ZCA from Zephyranthes candida, Amaryllidaceae (AAM94381). ZGA from Zephyranthes grandiflora, Amaryllidaceae (AAP37975); ASqA from Annona squamosa, Annonaceae (ABF 72850); AHA from Arisaema heterophyllum, Araceae (AAP50524); AKA from Amorphophallus konjac, Araceae (AAP22169); ALA from Arisaema lobatum, Araceae (AAS66304); AMA from Arum maculatum, Araceae (AAC48997); APaA from Amorphophallus paeonifolius
Araceae (ACA96190); CEA from Colocasia esculenta, Araceae (BAA03722); LRA from Lycoris radiate, Amaryllidaceae (BAD 98797); NHA from Narcissus hybrid, Amaryllidaceae (AAA33546); PPA from Pinellia pedatisecta, Araceae (AAR27793); PTA from Pinellia ternata, Araceae (AAP20876); TDA from Typhonium divaricatum, Araceae (AAQ55289); ACoA from Ananas comosus, Bromeliaceae (AAM28277); DPA from Dioscorea polystachya, Dioscoreaceae (BAD67184); HMA from Hernadia moerenhoutiana, Hernandiaceae (AAD45250); HHA from Hyacinthoides hispanica, Hyacinthaceae (AAD16403); CSA from Crocus sativus, Iridaceae (AAK29077); CVA from Crocus vernus Iridaceae (AAG10402); THA from Tulipa hybrid cultivar, Liliaceae (S62647); DFA from Dendrobium findleyanum, Orchidaceae (ABU62812), DOA from Dendrobium officinale, Orchidaceae (AAV66418); EHA from Epipactis helleborine, Orchidaceae (AAC48927); PCA from Polygonum cyrtonema, Ruscaceae (AAM28644); PCyA from Polygonatum cyrtonema, Ruscaceae (AAM77364), PMA from Polygonatum multiflorum, Ruscaceae (AAC49413); and PRA from Polygonatum roseum, Ruscaceae (AAW82332)
High hydrophobic residues in signal peptide region in pre-proprotein was similar to the signal peptides in other reported lectins [3]. The presence of sequence TGT at C- terminal region suggested the possible cleavage between
G and T as reported for other lectins [7, 25, 31]. C-terminal peptide helps in translocation of lectin to vacuoles [12]. Sequence analysis with BLAST-P revealed that, AAA belonged to single domain mannose binding lectin family
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Fig. 6 Expression analysis of aaa. Semi-quantitative RT-PCR was performed with RNA prepared from root, stem, bulb, Leaf, scape and flower for 15 cycles. Amplification of actin for 10 cycles served as internal control. The order of expression level was scape = flower [ bulb [ leaf. There was no expression in root and stem
Fig. 7 Expression of AAA in fusion with SUMO in E. coli. a Protein samples resolved on 15% SDS–PAGE, lane M molecular weight marker, lane 1 uninduced whole cell lysate (control), lane 2 induced whole cell lysate after 1 h, lane 3 after 2 h, lane 4 after 4 h, lane 5 supernatant, lane 6 AAA purified on HiTrap QXL. b Chromatogram showing purification of recombinant AAA on HiTrap QXL column, c Protein eluted in peak 1, confirmed by immuno-blot analysis
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and grouped together with A. cepa agglutinin (ACA), A. triquetrum agglutinin, (ATA) and A. porrum agglutinin (APA). It showed little distance from ASAL, A. sativum agglutinin (ASA) and A. ursinum agglutinin (AUA). NCBI conserved domain search suggested that AAA belonged to bulb type lectins with three conserved carbohydrate binding motifs. The motifs of AAA were different from ASAL by the presence of Gln (Q) in place of Asp (D) in 1st, Ala (A) in place of Arg (R) in 2nd and Glu (E) in place of Lys (K) in 3rd motif. It has been shown that the nature of amino acid residues in carbohydrate binding motif and their interaction in vicinity play important role in determining the affinity with ligands [23]. Besides differences in glycan binding motifs, ten other amino acid substitutions were also present (Fig. 4a). Some substitutions like N ? K, V ? R and D ? V could be important for unique biological properties of AAA. However, it needs to be established by reversion substitution study. Random coil and extended strand constituted interlaced domain of the main part of secondary structure, while ahelix was the core constituent of N-terminal signal peptide as reported for other bulb lectins [15]. Predicted tertiary structure appeared as a tight structural scaffold in which bsheets were predominant and connected with several turns and loops. The structure possessed three internal repeats, which formed beta prism like architecture. This was in good agreement with other reported B-lectins [6, 11, 15]. Like other mannose binding lectins, AAA expressed in a tissue specific manner at different levels, in different parts of the A. altaicum plant [6, 15]. We expressed AAA in fusion with SUMO in E. coli, although we could not achieve high expression as reported for SUMO-ASAL [29]. Further, we could not use (His)6 tag for the purification of the recombinant protein. We assume that the tag got buried in the protein scaffold and became inaccessible for binding to Ni–NTA column. Our attempt for purification of AAA by anion exchange chromatography was successful and we could purify the protein partially. Hemagglutination assay established biological activity in recombinant protein. Although it decreased as compared to native AAA, still It was 200-fold higher than ASAL [25, 29]. Screening of plant lectins from different sources and their characterization for various biological activities continues to remain an important area. Our project aimed to identify a lectin with higher insecticidal activity, although we found a new lectin with lower insecticidal but very high hemagglutination activity. Identification of the surface proteins in rabbit RBCs and other mammalian tissues with high affinity towards AAA and other lectins will be taken up in future. Acknowledgments This work has been funded by Council of Scientific and Industrial Research (CSIR), Government of India, Supra
Purification and Characterization of Allium altaicum Agglutinin Institutional Projects (SIP005). SKU, SS, RS and NKD acknowledge CSIR and PR acknowledge Indian Council of Medical Research, Government of India for respective Senior Research Fellowship. RT is thankful to DST for JC bose fellowship. The authors are also thankful to Moti Lal, SMH Abidi, Rajesh Srivastava and Aquila Bano for technical support.
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