Allelic differentiation of Kunitz trypsin inhibitor in wild soybean (Glycine soja)

Soybean Kunitz trypsin inhibitor (SKTI) has several polymorphic types, which are controlled by co-dominant multiple alleles at a single locus. Of these types, Tia and Tib are predominant types, and there are nine differences in amino acids between Tia and Tib. Recently, an intermediate transitional type (Tib ( i5 )) between them was detected. However, other transitional types have not been detected despite surveys of many cultivated and wild soybeans. One of the reasons why other transitional variants have not been found is inferred to be due to the difficulty of the detection of SKTI protein variants by polyacrylamide gel electrophoresis (PAGE). To detect novel variants of SKTI, nucleotide sequence analysis in addition to PAGE was carried out. Four new variants were found from many Japanese wild soybeans. Of these variants, three (designated as Tia ( a1 ), Tia ( a2 ), Tia ( b1 )) were detected through gene sequence analysis on wild soybeans having the same electrophoretic mobility as Tia, and one (Tig) was detected through PAGE. The Tig variant showed a slightly lower electrophoretic mobility than Tic. The nucleotide sequences of Tig were identical to those of Tib except for one T --> C transitional mutation at position +340. The sequences of Tia ( a1 )and Tia ( a2 ) genes were identical to those of Tia with the exception of a G --> A mutation at position +376 and a T --> C mutation at +404, respectively. The sequence of Tia ( b1 ) differed from Tia by three nucleotides: C --> A at position +331, T --> C at +459 and A --> G at +484. Of the three nucleotide changes, two were common to Tia ( b1 ), Tib ( i5 ) and Tib, suggesting that Tia ( b1 ) is an intermediate transitional type between Tia and Tib. Our results suggest that Tib type has been differentiated through a series of mutations from Tia before the domestication of cultivated soybean.     

Entity evidence for differentiation between Tia and Tib types of soybean Kunitz trypsin inhibitor: detection of a novel transitional variant type between Tia and Tib in wild soybean (Glycine soja Sieb. & Zucc.)

Soybean Kunit trypsin inhibitor (SKTI) has several polymorphic types. Of these SKTI, there are large differences of nine amino acid substitutions between Tia and Tib. So far no transitional type between them has been found. A novel transitional intermediate variant between Tia and Tib was detected in 11 lines from 720 Japanese wild soybeans (Glycine soja Sieb. & Zucc.). This variant showed identical electrophoretic mobility to Tib in the Davis system polyacrylamide gel electrophoresis (PAGE), but higher electric points than other SKTI proteins (Tia, Tib, Tic) in isoelectric focusing PAGE. The genetic analysis of SKTI in F₂ seeds from a cross between the novel variant type and Tib showed that this variant type is inherited as codominant alleles in a multiple allelic system at an SKTI locus. This variant also showed inhibitory activity to trypsin. We propose the genetic symbol Ti b ⁱ⁵ for this novel variant. The sequence analysis of Tib ⁱ⁵ revealed that six nucleotides were different between Tib ⁱ⁵ and Tia, and the nucleotides of these mutated positions were identical to Tib. This causes substitution of five amino acids at the residue position 62 (Tyr→Phe), 74 (Ser→Arg), 114 (Met→Val), 120 (Leu→Ile) and 137 (Pro→Thr). These substitutive amino acids are completely in accord with the amino acids of Tib, showing that Tib ⁱ⁵ is an intermediate between Tia and Tib types. Tib ⁱ⁵ type is widely distributed throughout seven separate areas from northeast to southwest Japan with a 1.5% frequency of total materials examined. This indicated that Tib ⁱ⁵ type did not originate from a recent mutation event, but had spread in wild soybean from ancient times.     

Genetic characterization of a leaf margin necrosis mutant in wild annual soybean (Glycine soja)

A leaf margin necrosis mutant was observed in a wild annual soybean (Glycine soja Sieb. & Zucc.) population from South Korea. Genetic studies showed that it was controlled by a single recessive nuclear gene, designatedlmn. TheLmn locus segregated independently of theAp, Dial, Dia3, Idh2, Pgi1 andTi isozyme loci.     

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.     

中国野生大豆的遗传多样性和生态特异性分析

野生大豆（Glycine soja）是栽培大豆的祖先,为东亚特有种,大部分分布在中国。我们采用52对SSR引物和10个植物学性状,以遗传丰富度和Simpson多样性指数为指标,对来自中国3个地理生态区域涉及24个省区的196份野生大豆所构成的代表性样本的遗传变异进行了研究,以期从分子水平和表型水平两个层面上揭示中国野生大豆遗传多样性和地理生态特异性。结果表明：中国野生大豆群体SSR位点的等位基因平均丰富度（NA）和平均Simpson多样性指数（H）分别为16.1和0.852,高于栽培大豆（NA=11.4,H=0.773）,野生群体的遗传多样性明显高于栽培群体。3个地理生态群体中南方群体多样性最高（NA=12.9,H=0.842）,黄淮海群体最低（NA=11.4,H=0.805）,东北群体居中（NA=12.5,H=0.834）。群体间存在遗传分化,不同群体具有不同的特异等位基因,位点AW132402（A2连锁群）、Satt522（F）、satt150（M）、Sat_332（D1a）、Satt046（K）、sct_190（K）等的一些等位基因只在特定群体出现,表现出群体分化后的生态特异性。中国野生大豆植物学性状的群体变异丰富,平均Simpson多样性指数为0.710。地理群体间存在分化,最明显的是生育期性状的分化,反映了地理、光照和温度等生态因子的选择作用,其中南方地理群体多样性最高（H=0.671）。SSR分子标记和植物学性状所获结果相对一致,表明中国野生大豆地理群体间性状分化有其遗传分化的基础。     

Stability of the allergenic soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.     

Reversible denaturation of the soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a beta-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70 degrees C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 degrees C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 degrees C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 degrees C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered beta-sheet proteins.     

Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]

The soybean Kunitz trypsin inhibitor (KTi) has several polymorphic variants. Of these, Tia and Tib, which differ by nine amino acids, are the two main types. In this study, differences in KTi proteome between Tia and Tib were investigated using three soybean cultivars and three mutant lines. Two cultivars, Baekwoon (BW) and Paldal (PD), and one mutant line, SW115-24, were Tia type, whereas one soybean cultivar, Suwon115 (SW115), and two mutant lines, BW-7-2 and PD-5-10, were Tib type. Protein from the six soybean lines was extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE), and two-dimensional polyacrylamide gel electrophoresis (2-DE). By SDS-PAGE, there was no difference between soybean cultivars and mutant lines, except for SW115-24. Western blot analysis revealed that, in comparison with Tia, Tib type accumulated relatively low amounts of KTi. By non-denaturing PAGE, the three soybean lines of Tib type were characterized by slower mobility than the three soybean lines of Tia type. Zymography detected eight distinct zones of trypsin inhibitory activity among which Tia and Tib lacked the fifth and sixth zone, respectively. By two-dimensional native polyacrylamide gel electrophoresis (2-DN), the spots related to trypsin inhibitory activity showed different mobilities, whereas only one KTi (21.5?kDa) spot was resolved by 2-DE. By two-dimensional zymography (2-DZ), Tib showed a broader activity zone (pI 4-7) in comparison with Tia (pI 4-5). The results indicate that the genotypes with a different type of KTi present different proteomic profiles and trypsin inhibitory activities.     

Three-fold structural pattern in the soybean trypsin inhibitor (Kunitz)

The molecule contains three very similar irregular Y-shaped lobes of antiparallel twisted β-sheet, which are grouped symmetrically round a central axis and linked by hydrogen bonds to form a six-stranded barrel. Each lobe can be superposed on either neighbour by a rotation of approximately 120 °. Of the 160 residues seen in the X-ray electron density map, 101 may be superposed onto other residues within a root-mean-square distance of 2.1 Å. The bond which reacts with trypsin lies on a loop between the first two lobes. It is suggested that the protein evolved from a primitive symmetrical trimer of identical subunits by tandem gene triplication.     

Proteomic and genomic characterization of Kunitz trypsin inhibitors in wild and cultivated soybean genotypes

In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.     

A circular dichroism study of the cyanogen bromide fragments of soybean trypsin inhibitor (Kunitz).

Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.     

Hydrogen-tritium exchange kinetics of soybean trypsin inhibitor (Kunitz). Solvent accessibility in the folded conformation.

The hydrogen exchange kinetics of Kunitz soybean trypsin inhibitor (STI) has been studied at pH 2, 3, and 6.5. From the temperature dependence of proton exchange at low pH, THE CONTRIBUTION OF MAJOR, REVERSIBLE PROTEIN UNFOLDING To the hydrogen exchange kinetics has been determined. Exchange directly from the folded conformation is characterized by an apparent activation energy (E*app) of approximately 25 kcal/mol, close to that of the chemical exchange step. At pH 6.5 the protein is more temperature stable than at low pH, and exchange of all but congruent to 8 protons can be observed to exchange with E*app congruent to 27 kcal/mol. This implies that all but congruent to 8 protons are accessible to exchange with solvent in the solution structure of folded STI. Estimates can be made of the average number of water molecules per molecule of STI consistent with a solvent accessibility model of hydrogen exchange kinetics. These estimates indicate that very few water molecules within the protein matrix are necessary to explain the exchange data. Calculations are done for the STI hydrogen exchange kinetics at pH 3, 30 degrees, approximating STI structure by a sphere of radius = 18 A. These calculations indicate an average of congruent to 4 water molecules in the shell from 13 to 16 A. from the center of the molecule, while less than 1 water molecule is indicated in the innermost 13 A. These calculations also suggest that there are congruent to 190 water molecules associated with the outermost 1.5-2 A of the sphere. While these values are consistent with a hydrophobic region in the central protein matrix, they indicate more solvent accessibility in the outer 1/3 of the molecule than the static accessibility estimates made from X-ray coordinates. Our results suggest that any protein movements or fluctuations responsible for solvent accessibility in proton exchange processes are localized in the outer regions of the globular structure.     

Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds

Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SP-Toyopearl 650M, Sephacryl S-200SF, and DEAE-Toyopearl 650S columns. Reverse-phase HPLC finally gave pure inhibitors. All of the inhibitors inhibited trypsin with dissociation constants of 3.2-6.2 x 10(-9) M. Some of the inhibitors inhibited chymotrypsin and elastase as well. Two inhibitors (VIIb and VIII) with a molecular weight of 20,000 were classified as a soybean Kunitz inhibitor family. Others (I-VIla) had a molecular weight of about 8,000, and were stable to heat and extreme pH, suggesting that these belonged to the Bowman-Birk inhibitor family. Partial amino acid sequences of four inhibitors were also analyzed. The complete sequence of inhibitor IV was ascertained from the nucleotide sequences of cDNA clones encoding isoinhibitors homologous to soybean C-II.     

Chemical-enzymatic insertion of an amino acid residue in the reactive site of soybean trypsin inhibitor (Kunitz).

Modified (Arg63-Ile64 reactive-site peptide bond hydrolyzed) soybean trypsin inhibitor (Kunitz) with all reactive amino groups, except that of Ile64, protected was described in the preceding paper (Kowalski, D., and Laskowski, M., Jr. (1976), Biochemistry, preceding paper in this issue). Treatment of this inhibitor with tert-butyloxycarbonyl-Ala- and tert-butyloxycarbonyl-Ile-N-hydroxy-succinimide esters yields inactive endo-tert-butyloxycarbonyl-Ala63A-and endo-tert-butyloxycarbonyl-Ile63A-modified inhibitors. The tert-butyloxycarbonyl groups were removed by treatment of the proteins with trifluoroacetic acid. After renaturation and purification, the resultant endo-Ala63A- and endo-Ile63A-modified inhibitors co-electrophorese with modified inhibitor both on disc gels (pH 9.4) and sodium dodecyl sulfate gels (after reduction of disulfide bonds) and show end groups corresponding to the 63A residue. These derivatives fail to form stable complexes with trypsin, extending the previous observation (Kowalski, D., and Laskowski, M., Jr. (1972), Biochemistry 11, 3451) that acylation of the P1' residue in modified inhibitors leads to inactivation. However, the incubation of endo-Ala63A- and endo-Ile63A-modified inhibitors with trypsin at pH 6.5 leads to the synthesis of the Arg63-Ala63A and Arg63-Ile63A peptide bonds in 4% yield. This is very close to the yield anticipated from a semiquantitative theory for the value of the equilibrium constant for reactive-site peptide bond. An alternative chemical method of insertion is also described. Controlled treatment of modified inhibitor with the N-carboxyanhydride of Glu produced inactive endo-Glu63A-modified inhibitor. Incubation of this inactive derivative with trypsin at pH 6.5 leads to 16% synthesis of the Arg63-Glu63A peptide bond. The higher yield of single chain protein in this case is attributed to the influence of the negative charge of the Glu63A side chain. Thus, the insertion of an amino acid residue between the P1 and P1' residues in soybean trypsin inhibitor (Kunitz) converts a trypsin inhibitor into a trypsin substrate.     

Cloning and functional characterization of an O-acetylserine(thiol)lyase-encoding gene in wild soybean (Glycine soja)

The terminal step of soybean cysteine synthesis is catalyzed by O-acetylserine(thiol)lyase (OAS-TL, EC 2.5.1.47). In this study, we isolated and characterized an OAS-TL gene from a wild soybean material (designated as GsOAS-TL1). GsOAS-TL1 cDNA sequence showed strict conservation at both nucleotide and amino acid levels compared with that from cultivated soybean. Genomic structure analysis of GsOAS-TL1 indicated that it contained 10 exons and 9 introns in the coding region with conserved exon sizes and intron locations compared with Arabidopsis thaliana OAS-TL-like genes. Among the complete GsOAS-TL1 cDNA and three part-deletion fragments, only expression of the full-length cDNA could rescue the NK3 cys⁻ Escherichia coli auxotroph, which was coherent with the assayed enzyme activity of purified fusion proteins. For RT-PCR analysis in different wild soybean tissues, GsOAS-TL1 showed lower expression in roots and developing seeds, whereas total OAS-TL activity of corresponding tissues showed significantly higher level in seeds than other tissues. To our knowledge, this is the first report on cloning and characterization of an OAS-TL gene from wild soybean. Our results are informative to further elucidate the function and evolution of OAS-TL in soybean.     

The possible origin of thick stem in Chinese wild soybean (Glycine soja)

Thicker, erect stem and enlarged seeds are characteristic of the domestication of cultivated soybeans (Glycine max) from its progenitor, wild soybean (G. soja). Wild soybeans have different stem thicknesses but the thick stem as defined here appears in a small number of small-seeded wild soybeans (≤2.0 g/100-seeds) in China. However, little attention has been paid to this phenomenon in considering the origin of thick stem in wild soybean. Here, we addressed this question through the study of a mixed field of wild, semi-wild and cultivated soybeans. Thick-stemmed samples had lower sensitivity to light period, higher mean genetic diversity (H _e = 0.090, H = 0.535) and higher mean multilocus outcrossing rate (t _m = 9.77 %), while thin-stemmed plants were the opposite (H _e = 0.029, H = 0.416) and lower mean outcrossing rate (t _m = 5.88 %). F statistics calculations indicated that there was genetic differentiation between the thin and thick stems. UPGM cluster analysis showed that not only were thick-stemmed wild soybeans genetically different from thin-stemmed ones, but they were also genetically closer to semi-wild soybean, to varying degrees completely dependent on seed size. These data strongly implied that the plants with thick stems had more complicated genetic backgrounds than the thin-stemmed ones, and that they were related to cultivated soybeans. This study suggests that if plants have distinctly thick stems (an average 2.5-fold thicker than other thin-stemmed plants) or stems similar to semi-wild plants and/or near to local soybeans in a natural wild population adjacent to farmlands, such plants could be cultivar-introgressive offspring.     

Expression of soybean Kunitz trypsin inhibitor gene SKTI in Dunaliella salina

Recently, microalgae have attracted much attention as a new bioreactor system for producing high-value heterologous proteins. In this paper, we investigated the expression of soybean Kunitz trypsin inhibitor gene SKTI in the chlorophyte Dunaliella salina. Using D. salina genomic DNA as template, the entire coding region of SKTI was amplified by PCR as a 654-bp DNA fragment. It had 100 % identity with the published sequence of SKTI. The entire SKTI fragment was cloned into the expression vector pCAM2201 at a site just downstream of 35S promoter of to yield pCAMSKTI. D. salina cells transfected with pCAMSKTI by means of the lithium acetate/polyethylene glycol-mediated method expressed a protein of 20.1 kDa as detected by anti-SKTI antibody. In addition, SDS-PAGE analysis of the cell extract also revealed an intense protein band indicative of the recombinant SKTI. SKTI was therefore successfully expressed in D. salina, and the expression could be detected for at least 35 generations.