Karl A. Wilson

Differential Expression of Soybean Cysteine Proteinase Inhibitor Genes during Development and in Response to Wounding and Methyl Jasmonate

Three cysteine proteinase inhibitor cDNA clones (pL1, pR1, and pN2) have been isolated from a soybean (Glycine max L. Merr.) embryo library. The proteins encoded by the clones are between 60 and 70% identical and contain the consensus QxVxG motif and W residue in the appropriate spatial context for interaction with the cysteine proteinase papain. L1, R1, and N2 mRNAs were differentially expressed in different organs of plants (juvenile and mature) and seedlings, although N2 mRNA was constitutive only in flowers. R1 and N2 transcripts were induced by wounding or methyl jasmonate (M-JA) treatment in local and systemic leaves coincident with increased papain inhibitory activity, indicating a role for R1 and N2 in plant defense. The L1 transcript was constitutively expressed in leaves and was induced slightly by M-JA treatment in roots. Unlike the chymotrypsin/trypsin proteinase inhibitor II gene (H. Pena-Cortes, J. Fisahn, L. Willmitzer [1995] Proc Natl Acad Sci USA 92: 4106–4113), expression of the soybean genes was only marginally induced by abscisic acid and only in certain tissues. Norbornadiene, a competitive inhibitor of ethylene binding, abolished the wounding or M-JA induction of R1 and N2 mRNAs but not the accumulation of the wound-inducible vspA transcript. Presumably, ethylene binding to its receptor is involved in the wound inducibility of R1 and N2 but not vspA mRNAs. Bacterial recombinant L1 and R1 proteins, expressed as glutathione S-transferase fusion proteins, exhibited substantial inhibitory activities against vicilin peptidohydrolase, the major thiol endopeptidase in mung bean seedlings. Recombinant R1 protein had much greater cysteine proteinase inhibitor activity than recombinant L1 protein, consistent with the wound inducibility of the R1 gene and its presumed role in plant defense.

Mobilization of seed protein reserves.

The mobilization of seed storage proteins upon seed imbibition and germination is a crucial process in the establishment of the seedling. Storage proteins fold compactly, presenting only a few vulnerable regions for initial proteolytic digestion. Evolutionarily related storage proteins have similar three-dimensional structure, and thus tend to be initially cleaved at similar sites. The initial cleavage makes possible subsequent rapid and extensive breakdown catalyzed by endo- and exopeptidases. The proteolytic enzymes that degrade the storage proteins during mobilization identified so far are mostly cysteine proteases, but also include serine, aspartic and metalloproteases. Plants often ensure early initiation of storage protein mobilization by depositing active proteases during seed maturation, in the very compartments where storage proteins are sequestered. Various means are used in such cases to prevent proteolytic attack until after imbibition of the seed with water. This constraint, however, is not always enforced as the dry seeds of some plant species contain proteolytic intermediates as a result of limited proteolysis of some storage proteins. Besides addressing fundamental questions in plant protein metabolism, studies of the mobilization of storage proteins will point out proteolytic events to avoid in large-scale production of cloned products in seeds. Conversely, proteolytic enzymes may be applied toward reduction of food allergens, many of which are seed storage proteins.

Characterization of a Soybean [beta]-Conglycinin-Degrading Protease Cleavage Site

Protease C1, an enzyme from soybean (Glycine max [L.] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the [alpha][prime] and [alpha] subunits, but not the [beta] subunit, of [beta]-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the [alpha][prime] and [alpha] subunits of [beta]-conglycinin may be located in their N-terminal domain, which is not found in the [beta] subunit of [beta]-conglycinin. To check this hypothesis, storage proteins from other plant species that are homologous to either the [alpha][prime]/[alpha] or the [beta] subunit of [beta]-conglycinin were tested as substrates. As expected, the convicilin from pea (Pisum sativum), a protein homologous to the [alpha][prime] and [alpha] subunits of [beta]-conglycinin, was digested by protease C1. The vicilins from pea as well as vicilins from adzuki bean (Vigna angularis), garden bean (Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), and mung bean (Vigna radiata), storage proteins that are homologous to the [beta] subunit of soybean [beta]-conglycinin, were not degraded by protease C1. Degradation of soybean [beta]-conglycinin involves a sequential attack of the [alpha] subunit at multiple sites, culminating in the formation of a stable intermediate of 53.5 kD and a final product of 48.0 kD. The cleavage sites resulting in this formation of the intermediates and final product were determined by N-terminal analysis. These were compared to the known amino acid sequences of the three [beta]-conglycinin subunits. Results showed these two polypeptides to be generated by proteolysis of the [alpha] subunit at regions bearing long strings of acidic amino acid residues.

Relevance of multiple soybean trypsin inhibitor forms to nutritional quality.

Trypsin inhibitors contribute to the antinutritional component of raw soybean meal by inhibiting vertebrate pancreatic serine proteinases in the small intestine, resulting in a range of deleterious physiological effects in the animal. The variation in the nutritional quality of soybean cultivars stems partly from wide-ranging levels of trypsin inhibitor, and from varying proportions of trypsin inhibitors of two classes--the Kunitz and the Bowman-Birk inhibitor classes. The latter class is better able to survive heat processing and digestion in the stomach. Some variation in cultivars also arises from the array of isoinhibitors present in the seed. The three Kunitz isoinhibitors, Ti(a), Ti(b) and Ti(c) differ by as much as 1000-fold in their interaction with bovine trypsin. The Bowman-Birk isoinhibitors differ not only in their extent of interaction with trypsin, but in their spectrum of inhibition of the other pancreatic enzymes, chymotrypsin and elastase. In this chapter, we look at twenty-two Bowman-Birk inhibitors from ten soybean cultivars and find at least twelve which are different enough in amino acid composition and/or inhibitor activity to be distinct protein species. Of these, three pairs are related by proteolytic digestion. Quite ironically, the Bowman-Birk inhibitors, and to some extent the Kunitz inhibitors, contribute to the nutritional quality of soybeans by virtue of their high cystine content which supplements the low or negligible amounts of sulfur-containing amino acids in the storage proteins that comprise the bulk of the protein reserve in the seed.

Proteolysis of kunitz soybean trypsin inhibitor during germination

Abstract During the germination and seedling growth of soybeans ( Glycine max ) a new form of the Kunitz soybean trypsin inhibitor (KSTI) appears in the cotyledons distinct from the KSTI of the quiescent seed. This inhibitor has been purified from the germinated seeds of soybean cultivar Amsoy 71 and cultivar Fiskeby V. These cultivars contain the Ti a and Ti i variants of KSTI respectively in the dry seed. The inhibitors were characterized by sodium dodecylsulphate-polyacrylamide gel electrophoresis, amino acid analysis, and partial sequence determination. The newly appearing form in Amsoy 71 (Ti a m ) was found to be identical to Ti a except for the loss of the five residue sequence at the carboxylterminus of Ti a . The Ti b variant of KSTI was found to contain at least 199 amino acid residues (as opposed to 181 for Ti a ). The amino-terminal sequence of Ti b was found to be identical to that of Ti a for the first ten residues. The KSTI species appearing in Fiskeby V with germination, Ti b m , appears to be derived from Ti b by the loss of the carboxylterminal decapeptide.

Electrophoretic transfer protein zymography

Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS-PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored.

Degradation of trypsin inhibitors during soybean germination

Abstract The principal Bowman-Birk soybean trypsin inhibitor in the soybean seed, BBSTI-E, is converted to BBSTI-D by limited proteolytic removal of the last two amino acid residues at the carboxyl terminus. The enzyme that catalyses this activity, Protease B1, works at an optimum pH of 4, first appears at day one and peaks at day four. The data also suggest that a second enzyme, Protease B2, appears by day 9 and catalyses extensive proteolysis of both BBSTI-E and BBSTI-D, the latter at a much faster rate. Three separate activities, measured at pH 7 in the presence of sulphydryl reducing agents, with peaks at days one, three and eight, catalyse extensive proteolysis of BBSTI-D, BBSTI-E and of BBSTI-E and -D, respectively. Immunostained Western blots show that other Bowman-Birk isoinhibitors are also digested during germination and early growth.

Proteolysis of soybean Bowman-Birk trypsin inhibitor during germination

Abstract The Bowman—Birk type trypsin inhibitor, BBSTI-D, which appears in the cotyledons of germinated soybeans ( Glycine max ), was isolated in homogeneous form. BBSTI-D has an amino acid composition identical to the native Bowman—Birk soybean trypsin inhibitor (BBSTI-E) except for the loss of one glutamyl/glutaminyl residue and one aspartyl/asparaginyl residue. The amino-terminal sequence of BBSTI-D was identical to that of BBSTI-E. These data, as well as the compositions of the tryptic peptides from reduced carboxymethylated BBSTI-D, indicate that BBSTI-D is derived from BBSTI-E by the loss of the carboxyl-terminal residues Glu 70 —Asn 71 .

Nature of proteinase inhibitors released from soybeans during imbibition and germination

Abstract Proteinase inhibitors are released to a smaller extent from soybeans which have been pre-equilibrated to an atmosphere of high relative humidity (r.h.) compared to those equilibrated to low r.h. Seeds pre-equilibrated to high r.h. also exhibited better germination. Regardless of the states of seed hydration, both Kunitz and Bowman-Birk soybean trypsin inhibitors are released in parallel with respect to each other and to other proteins at germination times up to 100 hr. After 48 hr of germination, new forms of trypsin inhibitor appear in the leachate which react with anti-Bowman-Birk trypsin inhibitor antibodies. No new forms of Kunitz trypsin inhibitor were observed immunochemically during the first 100 hr of germination.

An assay for activity of arogenate dehydratase based upon the selective oxidation of arogenate

Abstract An improved method of assay is presented for arogenate dehydratase, an enzyme catalyzing the formation of l -phenylalanine from l -arogenate. The improvement consists of the inclusion of a step in which arogenate is selectively oxidized using potassium permanganate prior to the measurement of phenylalanine. Following removal of excess KMnO 4 , the phenylalanine present is measured fluorometrically under optimal conditions. A convenient qualitative test for the completeness of arogenate oxidation in the presence of other molecules which are oxidized by KMnO 4 is described. A rigorous evaluation of phenylalanine measurement by the method reported here was accomplished by comparison with three other methods, including amino acid analysis.