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• W2035564145 abstract "Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, is a crop native to Southeast Asia and tropical Africa. The seeds contain multiple forms of Bowman-Birk type inhibitors. The major inhibitor HGI-III, from the native seed with 76 amino acid residues exists as a dimer. The amino acid sequence of three isoforms of Bowman-Birk inhibitor from germinated horsegram, designated as HGGI-I, HGGI-II, and HGGI-III, have been obtained by sequential Edman analyses of the pyridylethylated inhibitors and peptides derived therefrom by enzymatic and chemical cleavage. The HGGIs are monomers, comprising of 66, 65, and 60 amino acid residues, respectively. HGGI-III from the germinated seed differs from the native seed inhibitor in the physiological deletion of a dodecapeptide at the amino terminus and a tetrapeptide, -SHDD, at the carboxyl terminus. The study of the state of association of HGI-III, by size-exclusion chromatography and SDS-PAGE in the presence of 1 mm ZnCl2, has revealed the role of charged interactions in the monomer ↔ dimer equilibria. Chemical modification studies of Lys and Arg have confirmed the role of charge interactions in the above equilibria. These results support the premise that a unique interaction, which stabilizes the dimer, is the cause of self-association in the inhibitors. This interaction in HGI-III involves the ϵ-amino group of the Lys24 (P1 residue) at the first reactive site of one monomer and the carboxyl of an Asp76 at the carboxyl terminus of the second monomer. Identification of the role of these individual amino acids in the structure and stability of the dimer was accomplished by chemical modifications, multiple sequence alignment of legume Bowman-Birk inhibitors, and homology modeling. The state of association may also influence the physiological and functional role of these inhibitors. Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, is a crop native to Southeast Asia and tropical Africa. The seeds contain multiple forms of Bowman-Birk type inhibitors. The major inhibitor HGI-III, from the native seed with 76 amino acid residues exists as a dimer. The amino acid sequence of three isoforms of Bowman-Birk inhibitor from germinated horsegram, designated as HGGI-I, HGGI-II, and HGGI-III, have been obtained by sequential Edman analyses of the pyridylethylated inhibitors and peptides derived therefrom by enzymatic and chemical cleavage. The HGGIs are monomers, comprising of 66, 65, and 60 amino acid residues, respectively. HGGI-III from the germinated seed differs from the native seed inhibitor in the physiological deletion of a dodecapeptide at the amino terminus and a tetrapeptide, -SHDD, at the carboxyl terminus. The study of the state of association of HGI-III, by size-exclusion chromatography and SDS-PAGE in the presence of 1 mm ZnCl2, has revealed the role of charged interactions in the monomer ↔ dimer equilibria. Chemical modification studies of Lys and Arg have confirmed the role of charge interactions in the above equilibria. These results support the premise that a unique interaction, which stabilizes the dimer, is the cause of self-association in the inhibitors. This interaction in HGI-III involves the ϵ-amino group of the Lys24 (P1 residue) at the first reactive site of one monomer and the carboxyl of an Asp76 at the carboxyl terminus of the second monomer. Identification of the role of these individual amino acids in the structure and stability of the dimer was accomplished by chemical modifications, multiple sequence alignment of legume Bowman-Birk inhibitors, and homology modeling. The state of association may also influence the physiological and functional role of these inhibitors. Bowman-Birk inhibitors (BBIs) 1The abbreviations used are: BBI, Bowman-Birk inhibitor; HGI, horsegram inhibitor; HGGI, horsegram germinated inhibitor; HPLC, high performance liquid chromatography; GuHCl, guanidium hydrochloride; BAPNA, α-N-benzoyl-dl-arginine-p-nitroanilide HCl; DEPC, diethylpyrocarbonate; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; MS, mass spectrometry; T, total acrylamide concentration; C, degree of cross-linking.1The abbreviations used are: BBI, Bowman-Birk inhibitor; HGI, horsegram inhibitor; HGGI, horsegram germinated inhibitor; HPLC, high performance liquid chromatography; GuHCl, guanidium hydrochloride; BAPNA, α-N-benzoyl-dl-arginine-p-nitroanilide HCl; DEPC, diethylpyrocarbonate; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; MS, mass spectrometry; T, total acrylamide concentration; C, degree of cross-linking. are small serine proteinase inhibitors found in the seeds of legumes in particular (1Laskowski Jr., M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1928) Google Scholar). Characteristically, their molecular masses are in the range of 6–9 kDa. They are single polypeptides and comprise a binary arrangement of two sub-domains with a conserved array of seven disulfide bridges, which play a prominent role in the stabilization of their reactive site configuration (2Birk Y. Int. J. Peptide Protein Res. 1985; 25: 113-131Crossref PubMed Scopus (345) Google Scholar, 3Ikenaka T. Norioka S. Barret A.J. Salvenson G. Proteinase inhibitors. Elsevier, Amsterdam1986: 361-374Google Scholar). These inhibitors interact, simultaneously and independently with two (not necessarily identical) molecules of proteinases (4Harry J.B. Steiner R.F. Eur. J. Biochem. 1969; 16: 174-179Crossref Scopus (26) Google Scholar) without any conformational change (5Sierra I. Li de La Quillien L. Flecker P. Gueduen J. Brunie S. J. Mol. Biol. 1999; 285: 1195-1207Crossref PubMed Scopus (76) Google Scholar). The BBIs have two tandem homology regions comprising a consensus motif of three β-strands, each with a kinetically independent reactive site on the outermost exposed loop that adopts a common canonical conformation, similar to that of a productively bound substrate (1Laskowski Jr., M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1928) Google Scholar).In addition to protease inhibitory activity, the anticarcinogenic activity and radioprotective activity of BBIs from legumes have been widely studied (6Kennedy A.R. Am. J. Clin. Nutr. 1998; 68 (suppl.): 1406S-1412SCrossref PubMed Scopus (252) Google Scholar). Immune stimulating properties of these inhibitors have also been reported (7Harms-Ringdahl M. Forsberg J. Fedorcsak I. Ehrenberg L. Biochem. Biophys. Res. Commun. 1979; 86: 492-499Crossref PubMed Scopus (10) Google Scholar). The BBIs have been implicated to play a vital role in the arsenal defense mechanism that plants use to protect against insect predators and against environment hazards during germination and seedling growth.Despite extensive studies on BBIs, only a few three-dimensional structures have been solved by x-ray or by NMR. These include the x-ray structure of PI-II from tracy bean (8Chen P. Rose J. Love R. Wei C.H. Wang B.C. J. Biol. Chem. 1992; 267: 1990-1994Abstract Full Text PDF PubMed Google Scholar), A-II from peanut (9Suzuki A. Yamane T. Ashida T. Norioka S. Hara S. Ikenaka T. J. Mol. Biol. 1993; 234: 722-734Crossref PubMed Scopus (30) Google Scholar), and soybean BBI (10Werner M.H. Wemmer D.E. Biochemistry. 1992; 31: 999-1010Crossref PubMed Scopus (104) Google Scholar, 11Voss R.H. Ermler U. Essen L.O. Wenzl G. Kim Y.M. Flecker P. Eur. J. Biochem. 1996; 242: 122-131Crossref PubMed Scopus (100) Google Scholar), which have been analyzed in the free form, and PsTI-IVb from pea seeds (5Sierra I. Li de La Quillien L. Flecker P. Gueduen J. Brunie S. J. Mol. Biol. 1999; 285: 1195-1207Crossref PubMed Scopus (76) Google Scholar). The x-ray structure data of trypsin complexes with the BBIs from adzuki bean (12Tsunogae Y. Tanaka I. Yamane T. Kikkawa J. Ashida T. Ishikawa C. Watanabe K. Nakamura S. Takahashi K. J. Biochem. (Tokyo). 1986; 100: 1637-1646Crossref PubMed Scopus (84) Google Scholar), mung bean (13Lin G. Bode W. Huber R. Chi C. Engh R.A. Eur. J. Biochem. 1993; 212: 549-555Crossref PubMed Scopus (88) Google Scholar), and soybean (14Koepke J. Ermler U. Warkentin E. Wenzl G. Flecker P. J. Mol. Biol. 2000; 298: 477-491Crossref PubMed Scopus (71) Google Scholar) are available. A three-dimensional model of black-eyed pea BBI-chymotrypsin complex has been constructed based on the homology of BBIs (15de Freitas S.M. de Mello L.V. da Silva M.C. Vriend G. Neshich G. Ventura M. FEBS Lett. 1997; 409: 121-127Crossref PubMed Scopus (34) Google Scholar). The three-dimensional structure of a 16-kDa BBI from barley seeds at 1.9-Å resolution remains to be the highest resolution of a BBI to date (16Song H.K. Kim Y.S. Yang J.K. Moon J. Lee J.Y. Suh S.W. J. Mol. Biol. 1999; 293: 1133-1144Crossref PubMed Scopus (55) Google Scholar). The x-ray structure of a novel and unique monofunctional 14-amino acid residue cyclic peptide, from sunflower seeds, complexed with trypsin, has exhibited both sequence and conformational similarity to the trypsin-reactive site loop of BBIs (17Luckett S. Garcia R.S. Barker J.J. Konarev A.V. Shewry P.R. Clarke A.R. Brady R.L. J. Mol. Biol. 1999; 290: 525-533Crossref PubMed Scopus (317) Google Scholar).Horsegram (Dolichos biflorus), is a pulse crop native to Southeast Asia and tropical Africa. Four isoforms of BBIs, from horsegram seeds (18Sreerama Y.N. Das J.R. Rao D.R. Gowda L.R. J. Food Biochem. 1997; 21: 461-477Crossref Scopus (21) Google Scholar), have been isolated. The complete primary structure of the major isoform HGI-III has been determined (19Prakash B. Selvaraj S. Murthy M.R.N. Sreerama Y.N. Rao D.R. Gowda L.R. J. Mol. Evol. 1996; 42: 260-569Crossref Scopus (89) Google Scholar). Three linear epitopes of the major inhibitor have been mapped, of which one contains the chymotrypsin inhibitory site (20Sreerama Y.N. Gowda L.R. Biochim. Biophys. Acta. 1997; 1343: 235-242Crossref PubMed Scopus (10) Google Scholar). The role of disulfide linkages in maintaining the structural integrity of horsegram BBI was established by circular dichroism and fluorescence studies (21Ramasarma P.R. Appu Rao A.G. Rao D.R. Biochim. Biophys. Acta. 1995; 1248: 35-42Crossref PubMed Scopus (40) Google Scholar). Horsegram BBI followed the “two state” mode of unfolding and oxidative refolding of the BBI was possible only at very low inhibitor concentration in a disulfide-thiol buffer (22Singh R.R. Appu Rao A.G. Biochim. Biophys. Acta. 2002; 1597: 280-291Crossref PubMed Scopus (55) Google Scholar). The three new isoforms that appear upon germination of horsegram seeds (23Sreerama Y.N. Gowda L.R. J. Agric. Food Chem. 1998; 46: 2596-2600Crossref Scopus (3) Google Scholar) are derived from the dormant seed inhibitors by a limited proteolysis during germination and not by de novo synthesis (24Kumar P. Sreerama Y.N. Gowda L.R. Phytochemistry. 2002; 60: 581-588Crossref PubMed Scopus (22) Google Scholar). The inhibitors of horsegram (HGIs) are single polypeptides with a molecular mass of 8.5 kDa. SDS-PAGE and analytical gel filtration indicate the molecular mass to be ∼16 kDa (18Sreerama Y.N. Das J.R. Rao D.R. Gowda L.R. J. Food Biochem. 1997; 21: 461-477Crossref Scopus (21) Google Scholar), suggesting that they exist as dimers in solution. Such self-association and anomalous behavior on SDS-PAGE resulting in a large overestimation of molecular mass has been reported for several legume BBIs (25Wu C. Whitaker R. J. Agric. Food Chem. 1990; 38: 1523-1529Crossref Scopus (35) Google Scholar, 26Bergeron D. Neilson S.S. J. Agric. Food Chem. 1993; 41: 1544-1552Crossref Scopus (25) Google Scholar, 27Terada S. Fujimura S. Kino S. Kimato E. Biosci. Biotech. Biochem. 1994; 58: 371-375Crossref PubMed Scopus (45) Google Scholar, 28Godbole S.A. Krishna T.G. Bhatia C.R. J. Sci. Food Agric. 1994; 64: 87-93Crossref Scopus (51) Google Scholar). Many of the BBIs tend to undergo self-association to form homodimers or trimers or more complex oligomers (29Odani S. Ikenaka T. J. Biochem. (Tokyo). 1978; 83: 747-753Crossref PubMed Scopus (51) Google Scholar). The three-dimensional model of the blackeyed pea BBI-chymotrypsin complex (15de Freitas S.M. de Mello L.V. da Silva M.C. Vriend G. Neshich G. Ventura M. FEBS Lett. 1997; 409: 121-127Crossref PubMed Scopus (34) Google Scholar) and light scattering data (30Ventura M.M. Mizuta K. Ikemoto H. An. Acad. Bras. Cienc. 1981; 53: 195-201Google Scholar) suggest that the inhibitor molecules are in continuous equilibrium between monomers and several forms of multimers. The data available on the protein-protein interactions, responsible for the self-association of BBIs, is sparse. In contrast to the dry seed inhibitors (HGIs), the inhibitors of germinated horsegram seeds (HGGIs), derived from the dry seed inhibitors, are single polypeptides of ∼6.5–7.2 kDa and exist as monomers (24Kumar P. Sreerama Y.N. Gowda L.R. Phytochemistry. 2002; 60: 581-588Crossref PubMed Scopus (22) Google Scholar). In an attempt to understand and elucidate the structural features that contribute to the self-association of HGIs in solution, the primary structures of the HGGIs has been determined. The significant difference between the primary structures of inhibitors from the germinated seed (HG-GIs) and the inhibitor from the dormant seed (HGI-III) is the absence of the charged carboxyl-terminal tail and varied truncation at the amino terminus. This observation and the ability of HGI-III to self-associate and form dimers suggest that the structural elements responsible for this phenomenon occur at either the carboxyl and/or the amino terminus.The dimeric crystal structure of the pea seed BBI, PsTI-IVb (5Sierra I. Li de La Quillien L. Flecker P. Gueduen J. Brunie S. J. Mol. Biol. 1999; 285: 1195-1207Crossref PubMed Scopus (76) Google Scholar) reveals two monomers associated in a nearly perfect dimer that are mainly stabilized by an extensive hydrogen-bonded network, involving specific interactions between them, namely: (i) the guanidium group of Arg23 of one monomer and the polar group of side chain of Glu68 of second monomer and (ii) Lys16 of one monomer and the dyad-related carboxyl group of Glu69 of the other monomer. This observation and the finding that HGGIs, which lack the carboxyl-terminal Asp residues, lose the ability to form dimers suggest that these interactions play a unique role in the dimerization of BBIs. Based on these findings, chemical modification of the Lys/Arg residue, a comparative evaluation of the amino acid sequences of several BBIs that exist either as monomers or dimers and homology modeling of the dimer, we demonstrate the pivotal role of an interaction between Lys24 (trypsin reactive site) and Asp76 in HGI-III that characterizes the dimer formation. The effects of such dimerization on the functional aspects of the inhibitor are presented.EXPERIMENTAL PROCEDURESMaterials—Horsegram (D. biflorus) seeds were obtained locally. DEAE-Sephacel and Sepharose-4B were obtained from Amersham Biosciences. α-N-Benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA), cyanogen bromide, diethylpyrocarbonate (DEPC), bovine pancreatic trypsin (2 × crystallized, type III, EC 3.4.21.4), l-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin, endoproteinase Asp-N (EC 3.4.24.33), guanidine hydrochloride (GuHCl), 4-vinylpyridine, dithiothreitol, 1,2-dicyclohexanedione, and analytical gel filtration markers were procured from Sigma-Aldrich. Molecular weight markers for SDS-PAGE were from Bangalore Genei, Bangalore, India. All the other chemicals used were of highest purity.Purification of Isoinhibitors from Horsegram—Dormant seed inhibitor (HGI-III) and horsegram germinated inhibitors (HGGI-I, -II, and -III) were purified as reported earlier (18Sreerama Y.N. Das J.R. Rao D.R. Gowda L.R. J. Food Biochem. 1997; 21: 461-477Crossref Scopus (21) Google Scholar, 24Kumar P. Sreerama Y.N. Gowda L.R. Phytochemistry. 2002; 60: 581-588Crossref PubMed Scopus (22) Google Scholar).Trypsin Inhibitory Assay—The amidase activity of trypsin and its inhibition was assayed using the chromogenic substrate BAPNA as described earlier (24Kumar P. Sreerama Y.N. Gowda L.R. Phytochemistry. 2002; 60: 581-588Crossref PubMed Scopus (22) Google Scholar). One unit of trypsin enzyme activity is defined as the increase in the absorbance of 0.01 at 410 nm under the assay conditions. One inhibitory unit is defined as the amount of inhibitor, which reduces the enzyme activity by one unit.Polyacrylamide Gel Electrophoresis—Denaturing SDS-PAGE (15% and 17.5% T with 2.7% C) of HGGI-III and HGI-III was performed according to the procedure of Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). The gels were stained for protein with 0.1% Coomassie Brilliant Blue and destained.Protein Sequencing—Reduction and alkylation of HGGIs with 4-vinyl pyridine was carried out as described for the dry seed inhibitors earlier (18Sreerama Y.N. Das J.R. Rao D.R. Gowda L.R. J. Food Biochem. 1997; 21: 461-477Crossref Scopus (21) Google Scholar). The pyridylethylated HGGIs were cleaved with TPCK-trypsin and endoproteinase Asp-N according to Aitken et al. (32Aitken A. Geisow M.J. Findlay J.B.C. Holmes C. Yarwood A. Geisow M.J. Findlay J.B.C. Protein Sequencing a Practical Approach. IRL press, Oxford1989: 43Google Scholar). The pyridylethylated HGGIs were cleaved at the Met residue using a 50-fold molar excess of cyanogen bromide in formic acid over Met residues. Excess reagents were removed by repeated dilution and freeze-drying (33Allen G. Laboratory Techniques in Biochemistry and Molecular Biology: Sequencing of Proteins and Peptides. Elsevier, Amsterdam1989: 73Google Scholar). The lyophilized digests were dissolved in 0.1% trifluoroacetic acid, and peptides were purified by HPLC using a C-18 reverse phase column (Phenomenex ODS column, 250 × 4.6 mm, 5 μm) with 0.1% trifluoroacetic acid/CH3CN (7:3) linear gradient. The peptides were detected at 230 nm. The peak fractions were further rechromatographed using the same column and solvent system. The peptides were subjected to Edman analyses on an automated gas phase protein sequencer (Shimadzu PSQ-1).Size-exclusion Chromatography—Size-exclusion measurements were performed using a BIOSEP-SEC-S 3000 (300 × 8 mm, exclusion limit: 700 kDa for globular proteins) column on a Waters Associate HPLC equipped with a binary gradient pumping system and Waters Model 1296 photodiode array detector. The column was pre-equilibrated with the corresponding buffers prior to sample loading. The column was calibrated using a mixture of standard proteins, alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (14.4 kDa), and HGGI-III (6.4 kDa).SDS-PAGE analysis of Zn2+-induced HGI-III Monomerization—The inhibitor was incubated in the presence of 1 mm ZnSO4 for 1 h. The sample was then boiled with sample buffer for 5 min and separated by SDS-PAGE (17.5% T, 2.7% C) at pH 8.8. The separated proteins were then stained with 0.1% Coomassie Brilliant Blue.Chemical Modification of HGI-III—Diethyl pyrocarbonate (DEPC) was used to modify His residues. DEPC solution was freshly prepared by dilution of the reagent in cold ethanol. The concentration of the stock was determined by reaction with 10 mm imidazole (34Miles E.W. Methods Enzymol. 1977; 47: 431-442Crossref PubMed Scopus (811) Google Scholar). For modification with DEPC, HGI-III was diluted in 0.1 m phosphate buffer, pH 7.25. At fixed time intervals, aliquots of DEPC were added to the mixture, and the formation of N-carbethoxyhistidine was monitored by the increase in absorbance at 240 nm, using a Shimadzu UV-1601 double beam spectrophotometer. The final concentration of DEPC ranged from 0 to 0.4 mm. The modified HGI-III was chromatographed on a BIOSEP-SEC-S 3000 column pre-equilibrated in phosphate buffer, pH 7.25.Arginine residues of HGI-III were modified by the reviewed method of Smith (35Smith E.L. Methods Enzymol. 1977; 47: 156-161Crossref PubMed Scopus (34) Google Scholar) using 1,2-cyclohexanedione. HGI-III was dissolved in 0.2 m sodium borate buffer, pH 9.0 (0.1 mg/ml), and reacted with 0.15 m 1,2-cyclohexanedione at 35 °C for 2 h. The reaction mixture was acidified using 30% acetic acid and dialyzed against 10 mm acetic acid to remove excess reagents. The sample was concentrated and evaluated by SDS-PAGE.Lysine residues of HGI-III were modified at 25 ± 2 °C using citraconic anhydride (36Dixon H.B.F. Perham R.N. Biochem. J. 1968; 109: 312-314Crossref PubMed Scopus (344) Google Scholar). HGI-III (1 mg/ml) was dissolved in water and the pH adjusted to 8.0. Citraconic anhydride (1 μl/mg of protein) was added to the solution and maintained at pH 8. The modified inhibitor was desalted and analyzed for the monomer-dimer status by SDS-PAGE as described earlier.Modeling of HGI-III Dimer—The sequences of BBIs from leguminous plants were obtained from the NCBI protein sequence data base and aligned using the ClustalW multiple alignment algorithm (37Higgins D. Thompson J. Gibson T. Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55190) Google Scholar). Of these, a crystal structure for the pea BBI inhibitor was available from the Protein Data Bank (PDB: 1PBI). The sequences of the monomeric HGGI-III and dimeric HGI-III were highly similar to that of the pea inhibitor, enabling building of their models by standard homology modeling techniques. Models of HGI-III and HGGI-III were built using the Biopolymer module in InsightII (Accelrys Inc.) and energy-minimized using DISCOVER. The quality of the structure was measured using PROCHECK (38Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar). A single residue insertion at position 37, which forms part of a loop, was observed in HGI-III and HGGI-III, with respect to the crystal structure template. An analysis of related crystal structures in PDB using DALI (39Holm L. Sander C. J. Mol. Biol. 1996; 233: 123-138Crossref Scopus (3551) Google Scholar) and Insight-II indicated that the structure of BBI proteinase inhibitor PI-II (1pi2) contained a similar insertion at the same position. The insertion in the loop is therefore modeled based on this structure. Visualization and analysis of the structures were carried out using Insight-II.Thermal Stability Studies—The purified inhibitors were dissolved in water and incubated at 95 ± 1 °C in a constant temperature water bath. Aliquots were removed at regular time intervals, immediately cooled on ice, and assayed for residual trypsin inhibitory activity as described earlier.RESULTSPrimary Structure of the HGGIs and Comparison of Their Sequences—The complete amino acid sequences of HGGI-I, -II, and -III comprising of 66, 65, and 60 residues, respectively, were obtained by automated sequencing analysis of the proteins and peptides generated by enzymatic and chemical cleavage (Fig. 1, data shown only for HGGI-III). The molecular mass calculated on the basis of the sequence of HGGI-I, -II, and -III are 7109, 6993, and 6464, respectively. These results are in close agreement to the molecular mass determined by matrix-assisted laser desorption ionization-MS, which were 7216.7, 7074.6, and 6493.5, respectively. The determined amino acid composition agrees with that of the deduced sequence (24Kumar P. Sreerama Y.N. Gowda L.R. Phytochemistry. 2002; 60: 581-588Crossref PubMed Scopus (22) Google Scholar).The alignment of the amino acid sequences of the HGGIs with the sequence of HGI-III, the major isoinhibitor present in the dry seed is shown in Fig. 2. The sequences of the HGGIs are identical to HGI-III sequence, except for the truncation at both the amino and carboxyl termini of the sequence. The three inhibitors from germinated seeds (HGGIs) differ from each other only at the amino terminus. The absence of the charged tetrapeptide, -SHDD, at the carboxyl terminus is common to all the three HGGIs. The 14 half-cysteine residues are conserved in the HGGIs, as is the case of all legume BBIs sequenced thus far. The trypsin reactive site with Lys and chymotrypsin reactive site with Phe as the P1 residue in HGI-III remain unaltered in the sequence of the HGGIs. These results indicate that the three inhibitors of the germinated seed are in situ proteolytic products of the dry seed inhibitor HGI-III.Fig. 2Comparison of the amino acid sequences of HGGIs with HGI-III. The arrows are between the P1 and P1′ residues of the reactive site.View Large Image Figure ViewerDownload (PPT)SDS-PAGE and Size-exclusion Chromatography—SDS-PAGE analysis of HGGI-III indicates it is a single polypeptide with a molecular mass of ∼6.5 kDa (Fig. 3). HGGI-I and HGGI-II also move as single polypeptides of ∼7.0-kDa molecular mass on SDS-PAGE (24Kumar P. Sreerama Y.N. Gowda L.R. Phytochemistry. 2002; 60: 581-588Crossref PubMed Scopus (22) Google Scholar). In contrast, HGI-III moves as a single polypeptide of ∼16.0-kDa molecular mass (Fig. 3, lane C). The exact molecular mass of HGI-III as determined by electrospray mass spectrometry (18Sreerama Y.N. Das J.R. Rao D.R. Gowda L.R. J. Food Biochem. 1997; 21: 461-477Crossref Scopus (21) Google Scholar) and by sequence (19Prakash B. Selvaraj S. Murthy M.R.N. Sreerama Y.N. Rao D.R. Gowda L.R. J. Mol. Evol. 1996; 42: 260-569Crossref Scopus (89) Google Scholar) is ∼8.0 kDa. These results suggest that the HGI-III in solution undergoes self-association to form a dimer. The reduced and alkylated HGI-III is a polypeptide of ∼8.0 kDa (18Sreerama Y.N. Das J.R. Rao D.R. Gowda L.R. J. Food Biochem. 1997; 21: 461-477Crossref Scopus (21) Google Scholar).Fig. 3SDS-PAGE (15% T, 2.7% C) of HGI-III and HGGI-III. Lane A, low molecular weight markers; lane B, HGGI-III; lane C, HGI-III; and lane D, high molecular weight markers.View Large Image Figure ViewerDownload (PPT)The monomer/dimer status of HGI-III and HGGI-III was further evaluated by size-exclusion HPLC on a BIOSEP-SEC-S 3000 column using 0.25 m Tris-HCl, pH 7.25. HGI-III was well separated from HGGI-III (Fig. 4, A and B). HGGI-III eluted later with a retention time of 22.92 min corresponding to a molecular mass of 6.5 kDa. HGI-III elutes at 20.26 min, which corresponds to a molecular mass of ∼16.0 kDa. These results provide further evidence that HGI-III in solution associates to form a dimer.Fig. 4Size-exclusion chromatography of HGI-III and HGGI-III. The samples were dissolved in different buffers and loaded on to a BIOSEP-SEC-S 3000 column pre-equilibrated with respective buffers and eluted at 0.5 ml/min. A, HGI-III (pH 7.25); B, HGGI-III (pH 7.25); C, HGI-III (1 mm ZnSO4, pH 7.25); D, HGI-III (1 mm ZnCl2, pH 7.25); and E, HGI-III (1 mm ZnCl2, pH 6.5).View Large Image Figure ViewerDownload (PPT)Rationale for Chemical Modification—A closer evaluation of the sequences shows that the most significant difference between HGI-III and HGGI-III, is the physiological deletion of the peptide -DHHQSTDEPSES and the tetrapeptide -SHDD at the amino and carboxyl termini, respectively. Hence, either the depleted amino and/or the carboxyl termini are involved in the self-association of HGI-III. HGI-I, yet another of the isoinhibitors present in the dry seed of horsegram, although truncated at the amino terminus, exists as a dimer in solution (18Sreerama Y.N. Das J.R. Rao D.R. Gowda L.R. J. Food Biochem. 1997; 21: 461-477Crossref Scopus (21) Google Scholar). These observations implicate that the residues of the deleted tetrapeptide play a vital role in the dimerization of HGI-III. PsTI-IVb, a BBI from Winter pea seeds, has been crystallized as a nearly perfect 2-fold symmetric dimer in the asymmetric subunit, which includes its carboxyl-terminal segment (5Sierra I. Li de La Quillien L. Flecker P. Gueduen J. Brunie S. J. Mol. Biol. 1999; 285: 1195-1207Crossref PubMed Scopus (76) Google Scholar). The carboxyl-terminal tail from residues 68 to 70 (EEV), which constitutes an extended β-strand, makes no contact with its own subunit yet is held by interactions with the other subunit. Two specific interactions that have been discerned between the two subunits are (a) a hydrogen bond between the guanidium group of Arg23 of one subunit and the polar group of the side chain of Glu68 and (b) an ion pair between Lys16 of one subunit and the dyad-related carboxyl group of Glu69 of the other subunit. This observation, together with the fact that the deleted tetrapeptide contained Asp residues, suggests that such interactions could well be the premise to self-association in HGI-III. The effect of chemically modifying Arg and Lys residues of HGI-III has been studied. Chemical modification of these residues may disrupt such a subunit interaction in HGI-III leading to the formation of monomers.Chemical Modification of Arg and Lys Residues of HGI-III— The Lys residues of HGI-III were chemically modified using citraconic anhydride. Citraconylation resulted in the acetylation of the free ϵ-amino group of Lys. SDS-PAGE of the modified HGI-III revealed an increased relative mobility compared with the unmodified inhibitor (Fig. 5, lane D). The molecular mass, calculated on the basis of the relative mobilities of a set of standard proteins, was ∼8.5 kDa, corresponding to that of a monomer. The conversion of the dimer form of the HGI-III to monomer by citraconylation suggests that a Lys residue is involved in the self-association of HGI-III. The guanidium group of Arg residues were modified using 1,2-cyclohexanedione, resulting in a heterocyclic condensation product between the guanidium group of Arg and the carbonyl of 1,2-cyclohexanedione. SDS" @default.
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• W2035564145 date "2004-07-01" @default.
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• W2035564145 title "Molecular Mechanism of Dimerization of Bowman-Birk Inhibitors" @default.
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• W2035564145 doi "https://doi.org/10.1074/jbc.m402972200" @default.
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