Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry
2015; Wiley; Volume: 67; Issue: 12 Linguagem: Inglês
10.1002/iub.1451
ISSN1521-6551
AutoresGnanesh Kumar, P. Mishra, Vellareddy Anantharam, Avadhesha Surolia,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoIUBMB LifeVolume 67, Issue 12 p. 943-953 Research CommunicationFree Access Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry Gnanesh Kumar B S, Gnanesh Kumar B S Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaSearch for more papers by this authorPadmanabh Mishra, Padmanabh Mishra Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaSearch for more papers by this authorVellareddy Anantharam, Vellareddy Anantharam Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaSearch for more papers by this authorAvadhesha Surolia, Corresponding Author Avadhesha Surolia Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaAddress correspondence to: Avadhesha Surolia, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, Karnataka, India. Tel: +91-80-22932714. Fax: +91-80-23600535.E-mail: [email protected]Search for more papers by this author Gnanesh Kumar B S, Gnanesh Kumar B S Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaSearch for more papers by this authorPadmanabh Mishra, Padmanabh Mishra Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaSearch for more papers by this authorVellareddy Anantharam, Vellareddy Anantharam Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaSearch for more papers by this authorAvadhesha Surolia, Corresponding Author Avadhesha Surolia Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, IndiaAddress correspondence to: Avadhesha Surolia, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, Karnataka, India. Tel: +91-80-22932714. Fax: +91-80-23600535.E-mail: [email protected]Search for more papers by this author First published: 23 November 2015 https://doi.org/10.1002/iub.1451Citations: 9AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W102) in the activity of the lectin. © 2015 IUBMB Life, 67(12):943–953, 2015 Abbreviations ACN acetonitrile LAA Luffa acutangula agglutinin CIA Coccinia indica agglutinin ESI electrospray ionization MALDI-TOF-MS matrix-assisted laser desorption/ionization LC liquid chromatography CID collision-induced dissociation ETD electron transfer dissociation SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis. Introduction Lectins are a class of carbohydrate-binding proteins that bind monosaccharides, oligosaccharides, and glycoconjugates specifically and reversibly without enzymatically modifying them. Although they were first reported in plants, they have been found in all kingdoms of life ranging from viruses to animals. Because of the high sugar specificity, lectins have become indispensable tools for the past several decades in the analysis of glycoconjugates from various sources and in a variety of applications in immunological and histochemical studies 1. Phloem lectins constitute a specialized group of chitin-binding proteins 2-5. They were initially discovered in the fruits of plants belonging to Cucurbitaceae family and have become most widely studied members of this group. These lectins are also known as phloem protein 2 (PP2; ref. 6). In Cucurbita maxima, it has been noted that PP2 together with phloem protein 1 (PP1) constitute the main components of highly filamentous aggregates known as P-proteins 7. The presence of PP1 gene is limited to cucurbits, whereas PP2 is encoded in all angiosperms 8, 9. By virtue of their presence in phloem of plants and their interaction with chitooligosaccharides, they have been implicated to play a major role in plant defenses against fungi and bacteria 2 and in wound sealing 10. They may also aid in RNA binding and transport 11-13. Phloem lectin from Cucurbita maxima has been widely characterized 10, 14-16. The purification and further characterization of phloem lectins from other members of Cucurbits have also been reported from C. pepo 2, L. acutangula 3, S. edule 17, C. indica 4, and T. anguina 18. Two distinct forms of phloem lectins with molar mass 24,000–26,000 Da and 17,000 Da originally described from Surolia Laboratory are validated by gene sequencing to be present in many cucurbits 5. This indicates the species-specific differential expression of these phloem lectins. Biophysical studies by our group and others have shown that the phloem lectins exhibit high affinity toward chitotriose and higher oligomers but have low affinity for chitobiose 3, 4, 18. Despite the fact that few phloem lectins are biochemically and biophysically well characterized, no structural information at the atomic level is currently available for this class of lectin. Earlier Luffa acutangula agglutinin (LAA) was purified from the phloem exudates of ridge gourd on soybean agglutinin glycopeptide and characterized for its sugar specificity and biophysical properties with chitooligosaccharides 3. In the current study, we purified LAA by affinity chromatography on a chitin column and determined its primary structure using mass spectrometry. The chemical modification of tryptophan residues performed by N-bromosuccinimide showed loss of its hemagglutinating activity, which could be prevented when the modification is performed in the presence of chitooligosaccharides. A detailed analyses of the tryptophan modification by proteomics approach revealed that although three of the seven tryptophans per subunit in the lectin were accessible to oxidation by N-bromosuccinimide, only W102 could be protected by its ligand, N, N′, N″-triacetyl chitotriose. Materials and Methods Materials Ridge gourd was purchased from the local market. Chitin from shrimp shells, DTT, iodoacetamide, 2-mercaptoethanol, N-bromosuccinimide, sequence-grade trypsin, chymotrypsin, thermolysin, bovine serum albumin for gel filtration, and solvents (HPLC-grade purity) were purchased from Sigma (MO, USA). Chitooligosaccharides were obtained from Dextra Laboratories (Reading, UK), Precision Plus Protein™ Dual Color Standards, and Gel Filtration Standards were obtained from Bio-Rad (CA, USA). All other reagents used in this study were of high quality and procured from local suppliers. Methods Purification of Luffa acutangula Agglutinin on Chitin Affinity Matrix and Activity Assay The fresh ridge gourd fruits were washed in double-distilled water and bled with an incision of 1–2 mm depth. The exudate was harvested using ice-cold 20 mM PBS containing 10 mM 2-mercaptoethanol (buffer A). The extract was centrifuged at 3,900 × g (AG506R, Kubota 6500) for 30 min, and the clear supernatant was brought to 80% saturation by the addition of solid ammonium sulfate. The precipitate was separated by centrifugation at 15,620 × g (AG506R, Kubota 6500) for 30 min. All experiments were conducted at 4 °C unless stated otherwise. The pellet obtained was dissolved in buffer A and dialyzed extensively against the same buffer with multiple changes. The precipitate obtained during dialysis was discarded, and clear supernatant was applied on chitin matrix (150 mL; ref. 19) equilibrated with buffer A. The matrix was washed extensively with the same buffer until the absorbance at 280 nm (A280) was <0.05. The bound lectin was eluted with 20 mM acetic acid at room temperature. The protein was measured in each fraction by recording A280 values, and the fractions were pooled and dialyzed extensively against double-distilled deionized water containing 10 mM 2-mercaptoethanol. The activity of the lectin thus purified was checked by hemagglutination assays using 4% rabbit erythrocytes in 20 mM PBS. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis The purified lectin was analyzed on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions 20. The protein was stained with Coomassie brilliant blue R 250, and the gel was scanned on a Bio-Rad GelDoc. Size Exclusion Chromatography on Superdex 200 Dimeric state of the protein was determined by size exclusion chromatography using Superdex 200, 10/300 column (GE Healthcare). The column was first equilibrated with 10 mM PBS and calibrated with protein standards: γ-globulin (bovine), 158 kDa; bovine serum albumin, 66 kDa; ovalbumin (chicken), 44 kDa; myoglobin (horse), 17 kDa; and vitamin B12, 1.35 kDa. The void volume was determined by the position of the elution of thyroglobulin, and a standard of Ve/Vo was plotted against log(molecular weight). For the lectin sample, column was equilibrated with (10 mM) or without 2-mercaptoethanol followed by loading of 100 μL of protein sample (4 mg/mL) onto the column as separate runs, and then, the elution volume was noted. Protein Digestion with Different Proteases The peptides were prepared for de novo sequencing by subjecting the lectin to in-gel as well as in-solution proteolytic digestions using trypsin, chymotrypsin, and thermolysin as described in ref. 21. Intact Mass Analysis on Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Lectin was mixed with 1 µL of sinapinic acid matrix [0.1% trifluoroacetic acid/50% acetonitrile (ACN)] and spotted on matrix-assisted laser desorption/ionization (MALDI) target plate. The spectra were recorded using Bruker Daltonics ULTRAFLEX TOF-TOF instrument equipped with a smart beam laser and analyzed in linear mode using a 250 ns time delay and a 25 kV accelerating voltage in positive ion mode. External calibration was performed to a spectrum acquired for a mixture of proteins with masses ranging from 20,000 Da to 70,000 Da (Protein standard II, Bruker Daltonics). De Novo Sequencing Using LC-ESI-CID/ETD-MS/MS The peptides derived from the proteolytic digestions were subjected to liquid chromatography (LC; Agilent HP1100 Series), wherein the sample was passed through a reverse-phase column (Agilent Poroshell 120, SB C18, 4.6 mm × 150 mm, 2.7 µm) attached to an electrospray ionization (ESI) ion trap mass spectrometer. A linear-gradient elution was performed with H2O/ACN/0.1% formic acid for 60 min following at a flow rate of 0.2 mL/min. The eluting peptides were analyzed on the mass spectrometer, HCT Ultra PTM Discovery (Bruker Daltonics), which houses a classic ion trap (Paul type) with following parameters: nebulizer pressure of 30 psi, dry gas at a flow rate of 10 L/min, and dry temperature at 330 °C. For MS/MS experiments, the peptide precursor ions were fragmented by both collision-induced dissociation (CID) and electron transfer dissociation (ETD). Helium gas was used as the collision gas for CID experiments with MS/MS fragmentation amplitude of 1 V. ETD experiment was performed using fluoranthene as the electron transfer reagent and methane for the chemical ionization. The reaction time of electron transfer was typically set at 100 ms with the following parameters: maximum output of ETD reagent ion 202 m/z, scan range of 100–2,600 m/z, target mass of 900 m/z, and reagent ion change control was set to 200,000. The amino acid sequences were deduced by evaluating the fragment ion series (b- and y-/c-and z-type ions) observed in the corresponding MS/MS spectra processed on Data analysis software, version 4.1. The amino acids isoleucine (I) and leucine (L) are isobaric and are mostly assigned to as I or L based on appropriate database homology comparisons. Similarly, the assignment for glutamine (Q) and lysine (K) were made based on amino acids exhibiting an increment mass of 128 to Q based on appropriate database homology comparisons or K unless proven by a tryptic cleavage site 21. The protein sequence data reported in the current study will appear in the UniProt Knowledgebase under the accession number C0HJV2. Chemical Modification of Tryptophan with N-Bromosuccinimide Modification of tryptophan residues was carried out with N-bromosuccinimide according to Spande and Witkop 22. An aqueous solution of N-bromosuccinimide (10 mM) was added in 10 µL portions to a solution of lectin (1 mg/mL) in 10 mM sodium acetate buffer pH 4.0 at 25 °C, and the reaction was monitored spectrophotometrically using Varian CARY 100 Bio spectrophotometer. The blank titrations were also performed simultaneously by adding corresponding aliquot of N-bromosuccinimide to the reference cell. The reaction was stopped when the absorbance at 280 nm did not decrease further. The modified lectin sample was later dialyzed extensively against double-distilled deionized water in the presence of 2-mercaptoethanol, and the desalted sample was subjected to protease digestion [trypsin and trypsin/chymotrypsin (1:1)]. The modified tryptophan residues were identified as described above. Protection by Chitotriose Restricted portions (10 µL) of N-bromosuccinimide (10 mM) were added to lectin sample (1 mg/mL) in the presence of 5 mM of N, N′, N″-acetyl chitotriose, and the reaction was monitored spectrophotometrically as mentioned above. The hemagglutination assay was performed to estimate the loss in activity after the sample has been desalted. The lectin sample was also subjected to proteolytic digestion to map the tryptophan residues as mentioned above. Fluorescence Spectroscopy Fluorescence spectroscopic studies were performed on Jasco FP-6300 Spectrofluorometer for examining tryptophan emission intensity. An aliquot of the lectin solution (1 mg/mL) in 10 mM sodium acetate buffer pH 4.0 was excited at 295 nm while the emission was recorded from 310 to 400 nm. The excitation and emission slit widths were 5 nm each. Restricted portions (10 µL) of N-bromosuccinimide (10 mM) was added to the lectin sample until the fluorescence intensity reached near the baseline, and then, the average values of three consecutive scans were taken. Results Intact Mass and Primary Structure of Lectin Ridge gourd (Luffa acutangula) lectin was purified on chitin affinity matrix. The lectin migrated as a single protein band on SDS-PAGE under reducing and nonreducing conditions with the molar mass in the range of 24,000 ± 1,200 Da (Fig. 1A). On Superdex 200 size exclusion chromatography, the lectin eluted with a molar mass of 49,000 ± 2,000 Da and 48,000 ± 2,000 Da in the absence and presence of 2-mercaptoethanol, respectively (Supporting Information Fig. S1). The intact mass of the subunit as determined by MALDI-TOF-MS was 23,720 Da (+1 charge state; Fig. 1B). To obtain amino acid sequence of lectin, both the in-gel and in-solution proteolytic digestions were performed. De novo sequencing of peptides derived from these proteolytic cleavages gave rise to almost complete sequence of the protein that corresponds to 209 amino acids. Each amino acid has been determined from at least two different proteolytically derived peptides, and the primary sequence is shown in Fig. 1C. A homology search of the complete sequence using Blastp indicated that the lectin indeed belongs to phloem protein super family (Fig. 1D). Figure 1Open in figure viewerPowerPoint Intact mass and primary structure of LAA. A: SDS-PAGE (12%) analysis of purified lectin. Lane 1: purified lectin after treating with 10 mM DTT; lane 2: protein molecular weight markers; and lane 3: lectin without DTT treatment. B: MALDI-TOF mass spectrum of purified lectin. C: Amino acid sequence of LAA deduced from MS/MS spectra of peptide precursor ions derived from various proteolytic digests. D: Conserved domain database result showing that LAA belongs to the PP2 superfamily. Proteolytic digestion of lectin after reduction and alkylation using trypsin yielded a doubly charged precursor ions at m/z 738.5 and was subjected to CID experiments. The evaluation of almost complete b- and y-series ions clearly demonstrated the peptide sequence NAESNC(CAM)FMLYAR with carbamidomethyl cysteine (Fig. 2). This cysteine was further confirmed by the presence of propionamidomethyl derivative resulting in a 14-Da mass shift in the precursor ions at m/z 745.58 (Supporting Information Fig. S2). Additionally, doubly charged precursor ions were derived from chymotrypsin and thermolysin digestion at m/z 709.25 and 616.25 corresponding to WFDKNAESNC(CAM)F and FDKNAESNC(CAM)F peptide sequence, respectively, containing carbamidomethyl cysteine. A representative spectrum of ETD fragmentation (m/z 547.34+2) and the deduced peptide sequence VGHNLEAILK is shown in Supporting Information Figure S3. The amino acid sequence thus derived from the overlapping peptides obtained from various proteases was aligned with sequences of phloem lectins present in the database (NCBI) as shown in Fig. 3. Figure 2Open in figure viewerPowerPoint A: ESI ion trap fragment ion spectrum obtained from the doubly charged peptide precursor ions at m/z 738.5 derived from a tryptic digest after reduction and alkylation. B: Corresponding fragmentation scheme showing carbamidomethyl cysteine. Figure 3Open in figure viewerPowerPoint Multiple-sequence alignment of LAA with other cucurbits phloem lectin. C. digitata (gi|21952274), C. maxima (gi|1806), C. moschata (gi|8307830), C. argyrosperma (gi|508449), C. melo (gi|659131142), C. sativus (gi|571272670), and C. sativus (gi|449455417). The suspected residues involved in carbohydrate binding are underlined 23. Dashed lines represent gaps inserted for optimal alignment of the sequences. Modification of Tryptophan Residues To analyze the role of tryptophan in defining the activity of the lectin, the protein was subjected to tryptophan modification using N-bromosuccinimide. The lectin comprises seven tryptophan residues at W52, W73, W80, W82, W102, W135, and W165 in the sequence. The spectrophotometric monitoring revealed that the addition of 10 mM N-bromosuccinimide resulted in sequential modification of different tryptophan residues (Fig. 4), and six tryptophan residues/molecule are found to be oxidized as determined by the absorption measurement calculations according to Spande and Witkop 22. The loss in hemagglutinating activity against the number of tryptophan residues oxidized is shown in Fig. 4 (inset). Extrapolation of the graph on the ordinate shows that at least one tryptophan per subunit is involved in its carbohydrate-binding activity. The spectrofluorimetric analysis also showed a marked decrease in the fluorescence emission as the tryptophan residues are sequentially modified (Supporting Information Fig. S4). The modified lectin showed loss in activity up to 99% in the hemagglutination assay. Figure 4Open in figure viewerPowerPoint Effect of modification of tryptophan residues by N-bromosuccinimide on the absorption spectra of LAA. Native protein (control), 0.9, 1.5, 3.0, 4.2, and 5.9 residues per molecule were modified, respectively. Inset shows the effect of the extent of the modification of tryptophan residues on the hemagglutinating activity of LAA. The modified lectin was also subjected to proteolytic digestion to determine the extent and number of tryptophan residues oxidized. Both trypsin and trypsin/chymotrypsin digestion was performed to map the accessibility of the tryptophan residues in the protein. The analysis of fragment ion series of precursor ions revealed the presence of mono-oxidized (+16) and dioxidized (+32) form of tryptophan as shown in Figs. 5A and 5B for W73. Similarly, mono-oxidation and dioxidation were observed at W102 and W165. However, no oxidation could be identified on W52, W80, W82, and W135 (Table 1). Figure 5Open in figure viewerPowerPoint Mapping of modified W73. A and B: ESI ion trap fragment ion spectra of singly charged precursor ions at m/z 774.48 and at m/z 790.4, respectively, derived from trypsin/chymotrypsin digestion indicating mono-oxidation and dioxidation states. Table 1. Effect of the presence and absence of chitotriose on tryptophan residues during the modification of LAA by N-bromosuccinimide Tryptophan residue Protease used Oxidation Protection (+ chitotriose) W52 T ND ND W73 T/TC + − W80 T ND ND W82 TC ND ND W102 TC + + W135 TC ND ND W165 T/TC + – Abbreviations: T, trypsin; TC, trypsin/chymotrypsin; ND, not detected. Protection with Chitooligosaccharides When modification was carried out on the lectin in the presence of saturating concentrations of saccharides (5 mM), the loss in activity was in the range of only 20–25%, except in the case of chitobiose, the loss was calculated to be 65% (Table 2), indicating the enhancement in the protection in the presence of chitotriose and higher oligomers of N-acetyl-d-glucosamine. The modified protein in the presence of chitotriose was subjected to proteolytic digestion to determine the tryptophan residues that may resist modification. The evaluation of fragment ion series of a doubly charged precursor ion at m/z 944.40 derived from trypsin/chymotrypsin digestion revealed the amino acid sequence IDVCWLNIVGNIETSVL with W102 being unmodified (Fig. 6A). Modification in the presence of chitotriose partially protected W102 from oxidation as is evident from the decrease in the proportion of monoxygenated and dioxygenated tryptophan in this peptide (Fig. 6B). This confirmed that the W102 is the residue involved in its chitooligosaccharide-binding activity, and thereby, it was protected in the presence of chitotriose. The effect of N-bromosuccinimide on various tryptophan residues in LAA is summarized in Table 1. Figure 6Open in figure viewerPowerPoint Protection of W102 in the presence of chitotriose. A: ESI ion trap fragment ion spectrum of doubly charged precursor ions at m/z 944.40 derived from trypsin/chymotrypsin digestion indicating unmodified W102. B: Corresponding fragmentation scheme. C: ESI ion trap fragment ion spectrum of doubly charged precursor ions at m/z 960.40 derived from trypsin/chymotrypsin digestion indicating dioxidized W102. Inset shows the same peptide with mono-oxidized W102. Table 2. Effect of the presence of various saccharides during the modification of tryptophan residues of LAA by N-bromosuccinimide Saccharide (5 mM) Loss in hemagglutination (%) (GlcNAc)2 65 (GlcNAc)3 26 (GlcNAc)4 30 (GlcNAc)5 31 (GlcNAc)6 28 Discussion Phloem lectins are ubiquitously present in angiosperms but are well characterized from the Cucurbitaceae family plants only 2-4, 10, 15. The localization of these lectins in phloem of plants has led to the proposal on their involvement in plant defense, wound sealing, RNA transport, and so forth 11-13. A majority of these lectins have affinity toward chitooligosaccharides. Other than phloem lectins, a jasmonate methyl ester-inducible cytoplasmic/nuclear lectin Nictaba from Nicotiana tabacum has been found to interact with chitooligosaccharides 23. In the current study, LAA was purified from ridge gourd by affinity chromatography on chitin column to determine its primary structure. The results are consistent with our previous studies which suggested it to be a dimer consisting of two identical subunits of 24,000 Da, and the subunits are held together by noncovalent interactions (Fig. 1A and Supporting Information Fig. S1; ref. 3). The subunit nature of phloem lectins differs among Cucurbitaceae family, for example, pumpkin phloem lectin is a homodimer 10, whereas phloem lectin from snake gourd exists as heterodimer 18. Additionally, a minor peak at m/z 25,262 was observed and could be the possible precursor form of this lectin. Similar heterogeneity was also observed in pumpkin phloem lectin where the protein on SDS-PAGE at pH 9.2 could be resolved into two different bands with a small difference in their molar mass of 1,500 Da 10. A majority of the Cucurbitaceae phloem lectins sequence are derived either from cDNA or from genome sequence annotation 8, 24, 25. In many species, two distinct forms of lectins with molar mass of 24,000–26,000 Da 8, 10 and 17,000 Da 4 are present. The 17,000-Da lectin lacks up to 60 amino acid residues at its amino terminus when compared with the lectins in the mass range of 26,000 Da; however, it is composed of conserved domain structure and the lectin activity 8. In Luffa acutangula, 24,000-Da lectin is present in significant amount in the phloem (this study and ref. 3), and because of the nonavailability of its genome sequence, we determined its complete primary sequence of this lectin using mass spectrometry based on "bottom up" proteomics approach. The numbers of amino acids identified in LAA are 209 with two cysteine residues. The overall mass of 209 amino acids could be correlated well with the intact mass determined using MALDI-TOF-MS. The multiple-sequence alignment of the sequence thus determined for LAA with known phloem lectins from Cucurbitaceae family is shown in Fig. 3. The multiple-sequence alignment also depicts the residues suspected to be involved in the carbohydrate-binding specificity of this family of phloem lectins 16. The carbohydrate-binding site of a lectin contains amino acids with side chains that contribute to the association of a specific saccharide group, in a manner similar to enzyme–substrate association. The amino acid residues involved in sugar-binding activities can be determined either by chemical modification studies 26, 27 or by mutation of specific residues. In this study, the chemical modification of amino acid residues such as lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues of LAA did not alter its hemagglutinating activities (Supporting Information Table S1). However, the loss in activity was observed when the modification of tryptophan residue was carried out by N-bromosuccinimide. By absorption measurement calculations, it was found that six tryptophan residues per molecule were modified, and the hemagglutination activity implicated that one tryptophan residue per subunit is involved in the activity (Fig. 4). No tyrosine oxidation was observed in the absorption measurements in which the isosbestic point at 263 nm remains unaltered even after all accessible tryptophan residues are oxidized. The fluorescence emission intensity also decreased linearly and almost totally quenched due to the oxidation of all exposed tryptophan residues (Supporting Information Fig. S4). Similar studies on other lectins like Abrus agglutinin 28 and soybean agglutinin 29 have shown the involvement of at least one tryptophan residue per subunit in the lectin activity. However, the specific tryptophan residue involved in the activity has not been determined in this family of lectins. Recently, the mutation studies on pumpkin phloem lectin revealed that Ser104 is involved in the hemagglutinating activity and that the mutation of other residues like Leu100 and Lys200 also reduced the activity to a certain extent 30. In LAA and other few phloem lectins, cysteine was found to be present in place of serine (Fig. 3). To gain more insights on the nature of modification of tryptophan residues, the modified lectin was proteolyzed and subjected to LC-MS/MS. The proteolytic digestion with trypsin as well as trypsin/chymotrypsin together [as trypsin lacks cleavage site from amino acids (aa)82 to aa135] covered all seven tryptophan residues. The sequence of the peptide precursor ions comprising modified tryptophan showed both the mono-oxidation and dioxidation states (Fig. 5 and Table 1), and interestingly, the chymotrypsin has cleaved both the mono-oxidized and dioxidized tryptophan similar to unmodified residue (Fig. 5). Only modification of W73, W102, and W165 was observed and suggest that they are surface exposed. The other residues at W53, W80, W135, and W82 did not show any oxidation state, indicating their inaccessibility due to their burial in the interior of the protein. Thus, the N-bromosuccinimide tr
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