[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || The Protease Inhibitors of Seeds

9. The Protease Inhibitors of Seeds

KARL A. WILSONDepartment ofBiological Sciences , The State University ofNew Yorkat Binghamton ,Binghamton, NY 13902-6000, USA

ABSTRACT. Seeds commonly contain relatively high levels of protein proteinase inhibitors.The majority of the well characterized inhibitors are active against serine endopeptidases, whilea small number are inhibitors of cysteine endopeptidases. Sufficient amino acid sequence datahave accumulated to allow their grouping into nine families. Members of eight families inhib-it serine endopeptidases : the Kunitz Soybean Trypsin Inhibitor, the Bowman-Birk TrypsinInhibitor, the Squash Trypsin Inhibitor, the Potato Protease Inhibitor I, the CM Protein/NapinProtease Inhibitor, the Protein Z/Serpin, and the Maize Bifunctional Inhibitor/Thaumatin fam-ilies. One inhibitor family, the Phytocystatins, inhibits cysteine endopeptidases. Less wellcharacterized inhibitors of metalloproteinases have also been described, but their sequencedata are not yet available. The protease inhibitors appear to be involved primarily in defense ofthe seed from exogenous proteases such as those secreted by pathogenic microbes or by insectpests . In some cases the inhibitors may also act as storage depots for sulfur-containing aminoacids . During germination and early seedling growth, these inhibitors are degraded much likethe bulk storage proteins . In a relatively small number of instances the seed protease inhibitorsmay serve to regulate endogenous proteases. Transgenic plants expressing the appropriateseed protease inhibitors in aerial tissue have been found to have increased resistance to insectdepredation. This promises to be an effective way to control insect pests without the use ofenvironmentally damaging synthetic insecticides.

I. Introduction

In 1945 Kunitz made the observation that extracts of soybean seeds stronglyinhibited the digestion of casein by bovine trypsin. He subsequently purifiedthe trypsin inhibitory activity and showed that it was due to a discrete proteinspecies (Kunitz, 1945, 1946). Since this initial discovery, the seed proteaseinhibitors have remained a topic of continuing research . While the earlieststudies concentrated on inhibitors against mammalian serine endopeptidas-es, such as trypsin and chymotrypsin, we now know that seeds contain awide variety of inhibitors, including those active against cysteine endopep-tidases. Inhibitors of mammalian serine carboxypeptidases and of metallo-endopeptidases have also been described. The presence of protease inhibitorsappears to be a general character of plant seeds, and is observed in both themonocot and dicot lineages of the Angiospermae and in the Gymnospermae.

B.A. Larkins and IX. Vasil (eds.) . Cellular and Molecular Biology of Plant Seed Development. 331- 374.© 1997 Ktuwcr Academic Publishers.

332 Karl A. Wilson

Much of the research concerning the protease inhibitors has been directedat understanding their basic chemistry, struc ture, and physiology. However,this research has also addressed severalmore appli ed aspec ts of these proteins.

As Antinutrients - The relatively high concentration of protease inhibitorsin crop seeds consumed by humans and livestock suggests that the inhibitorsmay interfere with the digestion and hence utiliz ation of dietary proteins(Birk, 1985; Liener, 1975; Rackis et al., 1985; Liener et al., 1985; Spangleret aI., 1985; Gumbm an et aI., 1985).

As Models of Protein-Protein Interactions - The first well characterizedplant protease inhibitors, such as the Bowman-Birk and Kunitz soybeantrypsin inhibitors, proved to be excellent model s to study strong protein-protein interactions (Laskowski and Sealock , 1971; Finkenstadt et aI., 1974;Kowalski et al., 1974). Their relatively small size , formation of a stoichiomet-ric complex with the protease, and the ability to obtain sufficiently large , highquality crystals allowed the determination of crystal structure by X-ray dif-fraction techniques (Bode and Huber, 1992; Sweet et aI., 1974 ). The relativesimplicity of plant protease inhibitors also allowed the application of othertechniques for structure determination , such as NMR (Werner and Wemmer,1991,1 992; Baillargeon et aI., 1980).

As Potential Therapeutic Agents - Many aspects of normal body functionin mammals (and other animals) involve regulatory pathways involving prote-olytic events by specific proteases. There has been interest in the discovery ofplant protease inhibitors that spec ifica lly inhib it such regul atory proteolyticenzymes (e.g. as in the blood clotting/thrombolytic pathway), with an eye tobeing able to carefully regulate operation of the pathway under pathologicalconditions. Similarly, there is interest in the inhibitors as possible age nts in thecontrol of cellular proteases, such as cathepsin G and granulocyte ela stase, inthe inflammatory response (Hoj ima et aI., 1982; Meyer and van Staden, 1991;Hayashi et aI., 1994; Larionova et aI., 1994). Protease inhib itors have alsobeen implicated as anti-carcinogenic factors in the diet , another medicallyoriented research focus (Yavelow et aI., 1983; Troll et aI., 1987; Messina andBarnes, 1991).

As Protective Agents Against Herbivore Pests and Microbes - The findingthat some plant protease inhibitors are active again st the proteases of insectherbivores and microbial pathogens has implicated the inhibitors as possibl ecomponents in the resistance of plants to these pests.Th is has led to attempts togenetically engineer plants with higher pest resistance by creating tran sgenicplants that express high levels of inhibitors (Boulter et aI., 1989; Ryan , 1990;Chen et aI., 1992).The primary emphasis of this review will be the types, distribution, struc-

ture, and physiology of the protease inhibitors in plant seeds. Other aspects,such as those noted above, will be mentioned where appropr iate. The readeris also directed to previous reviews (Ryan, 1973; Richardson, 1977 ; Wilson,1981; Weder, 1981; Garcia-Olmedo et aI., 1987) that have been concerned

The Protease Inhibitors of Seeds 333

with seed protease inhibitors and plant protease inhibitors, either in generalor with regard to one of the aspects mentioned above.

II. The Classification of Protease Inhibitors

Initial studies of the seed protease inhibitors were primaril y concemed withinhibitors of the mammalian pancre atic serine proteases, such as trypsin,chymotrypsin , and , to a lesser ex tent, elastase. Thi s was undoubtedly due toa combination of the easy availability of these enzymes (either commerciallyor as prepared in the laboratory), simple assays for their activity, and theemphasis on the potential anti-nutritional qualiti es of the inhibitors. However,the increasing availability of other types of proteases, e.g. plant cysteineproteases (papain, bromelain, ficin) and variou s microbi al proteases (such assubtilisin), has led to the discovery of other types of inhibitors. Indeed, itappears likely that most, if not all , seed s contain a complex complement ofprotease inhibitors active against a number of types of proteases.The proliferation of inhibitor types originally made any rational classifica-

tion system difficult. But this situation has been simplified by the accumulationof sufficient data, parti cul arly in the form of amino acid sequences, to allowthe classification of the inhibitors into a number of discrete families based onsequence homology. Laskowski and Kato (1980) first proposed nine inhibitorfamilies based on amino acid sequence homology and disulfide-bonding pat-tern s. These famili es represented inhibitors found in plants, animals, andmicrobes. At the time, four of these inhibitor families were known fromplants. Further research , in particular additional sequence data , has sugges tedthe ex istence of at least 15 families (Bode and Huber, 1992, and this review).Nine of these have been observed in seeds. A summary of these families isgiven in Table I. While most known seed prote ase inhibitors can be assignedto one of the listed famili es, others are at present unassigned because ofinsufficient data, particularly primary structure data or at least amino acidcomposition and molecular mass data. The inhibitor families found in seedsare be examined below.

A. The Phenomenon of Isoinhibitors

A common observation in seeds is the presence of multiple inhibitor speciesactive against a particular type of protease (Weder, 1981; Hymowitz , 1983;Garcia -Olmedo, 1987). Multiple inhib itor forms that are active against thesame protease have been termed isoinhibitors. The presence of isoinhibitors oftrypsin and chymotrypsin is espec ially common. Isoinhibitors can sometimesarise due to the presence of inhibitors from two or more inhibitor familiesin the same seed (as in the soybean, where trypsin inhibitors from both theKunitz and Bowman-B irk famili es are present). Alternatively, there may be

334 Karl A. Wilson

TABLE I

The Fam ilies of Protease Inh ibitors Found in Plant Seeds

I.

2.

3.

4.

5.

6.

7.

8.

9.

Family

Kuni tz Soybean Try psin Inhibitor

Famil y

Bowman-Birk Inhibitor Family

Squash Tryp sin Inhibitor Family

Mustard Trypsin Inhibitor Family

Potato Proteinase Inh ibitor I Fami ly

CM Protein/Napin Protease Inhibitor Family

Protein Z/Serpin Family

Maize Bifunctional Inh ibitor/Thaumat in Famil y

Phytocystatin Famil y

Plant Famil ies Where Found

Leguminosae

Gramineae

Cucurbi taceae

Sterculi aceae

Leguminosae

Gram ine ae

Cucurbitaceae

Cucurbi taceae

Cruci ferae

Leguminosae

Gramineae

Cucurbitaceae

Amaranthaceae

Polygon aceae (?)

Cruc iferae

Gram ineae

Gramineae

G ram ineae

Leguminosae

Gram ineae

multiple forms present in the same family, arising either from multiple allelicforms of the same gene or multigene families encodin g similar yet distinctgene products (as in the multiple Bowman-Birk type inhibitor species foundin the soybean, see below). A third source of isoinhibitors is arti factual, i.e.one or more alternative species arise from the original inhibitor through par-tial proteolysis. This may be due to endogenous proteolytic enzymes whichcleave one or more peptide bonds in the inhibitor eith er in vivo, e.g. duringgermination (Wilson, 1988; Wilson and Chen, 1983), or in vitro during purifi-cation. A common experimental source of artifactual isoinhibitors is affinitychromatography. Affinity chromatography on immobilized bovine tryp sin isa common technique in the purification of trypsin inhibitors from seeds. Fol-lowing adsorption of the inhibitor to the affinity medium and washing atneutral or slightly alkaline pH, the bound inhibitor is eluted by a drop in pH.How this change in pH is accomplished determines the nature of the elutedinhibitor. Laskowski and coworkers have demonstrated that the rapid dissoci-ation of the trypsin-trypsin inhibitor complex favors the rele ase of the native


inhibitor, I . In contrast, a slower dissociation (favored by a slower drop inpH) favors the production of r' , the modified inhibitor, where a single peptidebond (located at the reactive site on the inhibitor which directl y interactswith the catalytic site of the protease) is cleaved. In the case of a trypsininhibitor, this is generall y a Lys-X or Arg-X peptide bond (Laskowski andSealock, 197I ; Liene r, 1975). Affinity chromatography can thus potentiallydoubl e the apparent number of inhibitor species present, giving rise to artifac-tual isoinhibitors (Swartz et aI., 1977; Lei and Reeck , 1986). This artifact ismost easily avoided by performing affinity chromatography with catalyticall yinactiv e anhydrotrypsin (Rayas-Duarte et al., 1992).

B. The Kunitz Soybean Trypsin Inhibitor Family

The archetype and namesake of this inhibitor family is the soybean trypsininhibitor first described by Kunitz in 1945 (Kunitz, 1945, 1946, 1947a,b).The Kunitz soybean trypsin inhibitor (KSTI) consists of a single polypep-tide chain of 181 amino acid residues (molecular mass 21.5 kDa). There arefour half-cystine residues in two disulfide bonds, Cys39-Cys86 and Cys136-Cys 145 (Koide et al., 1972; Koide and Ikenaka, 1973). KSTI has been shownto be localized primarily in the protein bodies and cell walls of the soybeancotyledon cells (Horisberge r and Tacchini-Vonlanthen ,1983b). The interac-tion of KSTI with bovine trypsin has been studied extensively by LaskowskiJr. and coworkers (Laskowski and Sealock , 1971; Finkenstadt et aI., 1974;Kowalski et aI., 1974; Baillargeon et aI., 1980), and has led to the reactive sitemodel. In this model the inhibitor forms a compl ex with the trypsin much asa substrate, but with the inhibitor a (relatively) stable, long-lived complex isformed rather than rapidl y dissociat ing to the free protease and two productpolypeptides. The inhibitor interacts with the trypsin molecule at the reactivesite of the inhibitor, a peptide bond which corresponds to the cleaved bondin a normal hydrolyzed substrate. Indeed, under the appropriate conditionsthe trypsin-trypsin inhibitor complex can dissociate to yield the protease andeither native ('virgin ' ) inhibitor, or the inhibitor with its reactive site bondcleaved (the 'modified ' inhibitor). The modified inhibitor is still active, andcan be converted back to the virgin form by reassociation with trypsin andsubsequent dissociation. Thi s model appears to fit the interaction of trypsin(and other related serine proteinases such as a -chymotrypsin and elastase)with a number of seed inhibitor type s. The KSTI and Bowman-Birk inhibitorfamilies (see below) have been best characterized in this regard . This mod-el of inhibitor/proteinase interaction is consistent with the structure of theKSTI-trypsin complex deduced by x-ray crystallography (Sweet et aI., 1974;Bode and Huber, 1992).The reactive site model pred icts that the reactive site bond should be

consistent with the cleav age specificity of the inhibited proteinase. In thecase of trypsin , the predicted reactive site bond , PI-PI ' (notation of Schecter

336 Karl A. Wilson

and Berger, 1967), should be either Arg-X or Lys-X. In fact, the reactivesite bond of KSTI is Arg63-Ile64 (Bidlingmeyer et aI., 1972). LaskowskiJr. and coworkers have demonstrated that replacement of the PI position ofthe reactive site bond of KSTI can drastically change the specificity of theinhibitor. While normal, Arg63-KSTI inhibits bovine trypsin strongly anda-chymotrypsin weakly (Kassocvalues of 1011 M- 1 and 3 x 105 M- I respec-tively), Trp63-KSTI is inactive as a trypsin inhibitor, yet strongly inhibitsa -chymotrypsin (Kassoc > 109 M- I ) (Kowalski et aI., 1974).Examination of many soybean strains and cross-strain crosses by Hymowitz

and coworkers (Clark et aI., 1970; Hymowitz and Hadley, 1972; Orf andHymowitz, 1979; Hymowitz, 1983) and by Freed and Ryan (1980) haverevealed that KSTI is coded for by a single locus that exists as three codom-inant alleles , Ti", Ti b, and Tic, and a recessive allele ti, where no functionalKSTI is synthesized. The Ti" to Tic inhibitor forms are readily distinguishableelectrophoretically. The Ti" form of KSTI is the ' classical' KSTI form andis found in most Chinese and American soybean varieties. The Ti b form ismost prevalent in soybean varietie s from Japan and Korea, while only a fewstrains expressing TiChave been identified (Clark et aI., 1970). The amino acidsequences of the KSTI variants have been examined by Kim et a1. (1985).All were found to have 181 amino acid residues. Ti'' and Ti b differed at 8positions in the sequence, while Ti" and TiCdiffered at only one residue. Incontrast, Hartl et al. (1986) found that Ti b isolated from the strain Fiskeby Vwas 18 residues longer than Ti". This suggests that there may in fact be twoTib forms that, while different in sequence, are identical electrophoretically.Kollipara and Hymowitz (1992) recently examined 11 wild perennial Glycinespecies by Western blotting . They found that most of these species containedmultiple forms (isoinhibitors) reacting with the anti-KSTI antibodies. Jofukuet al. (1989) have recently shown that the rece ssive allele ti results from aframeshift mutation that causes premature termination of transcription of theKSTI gene . The resulting truncated transcript is rapidly degraded, and henceno functional inhibitor protein is produced.KSTI was the only known member of this family for nearly 35 years.

Other common crop legumes, in particular those cultivated in Europe andthe Americas (e.g. Pisum, Phaseolus, Lens, Arachis, Vicia, etc.) all appearedto lack the Kunitz-type inhibitors, having instead the Bowman-Birk typetrypsin inhibitors (Norioka et aI., 1988). However, it has now become evidentthat this apparent uniqueness of soybean is a sampling artifact. Kunitz typeinhibitors have now been identified and characterized from a wide range oflegume genera, including Prosopsis (Negreiros et aI., 1991), Erythrina (Jou-bert, 1982a,b, 1986, 1988; Joubert and Sharon, 1985; Joubert et aI., 1985;Meyer and van Staden, 1991), Psophocarpus (Kortt et aI., 1983; Yamamotoet aI., 1983; Shibata et aI., 1988), Acacia (Joubert, 1983; Lin et aI., 1991),Adenanthera (Richardson et aI., 1986), Peltophorum (Joubert, 1981), Albizzia(Odani et aI., 1979), and Canavalia (Terada et aI., 1994a,b,c). These inhibitors

The Protease Inhibitors ofSeeds 337

The Kunitz Soybean Trypsin Inhibitor Family

KSTI - T I aWBTI -lweCt-)

ELT I - lPJTI - 6

BASITe T!

KST I -Ti aHAT ! - l

WBC I- JELTl -3PJTl -6

BA.SI1'eT I

K51' I -1'i awaT I - lwac I · )ELTI - )PJTI -6BAS I1'e T I

KSTI - T i ..WOT! - !

wa C I - )EL TI - )

PJTI- 6

BAS ITCT l

P'~K 1G fEi ~OrnD 'M-rnGW- - ill:~20•• v SDD' F:;0_-YKLVF e PQQl~O. D- fD1K~G --- -101p S v K L S l!lL K 5 T K F 0 'I L - - F K If E K V T 5 K F S S - • • Y K L K y eA K R • - • -Wor C K • l..QJP A V K L S O Q KLP E K D I L V - F KF E K V SH SNI H V - -YK L L YC QH DE E ~ D V K CO Q _ _ ·Y

L 5 V K L S [il o E ST Q F D Y P - - F K F E Q v S D K L H 5 - - - Y K L Ly e E G K - • - H EK e A - - - - 5

V K I A V KWO A R G F G P • - • - P R I R P H ROD · - • • - - y J( L V y eo E G Q K a-s illa C K - • • -mR RH VI T GP VKD P SP S()RE NAFR I EKYH G A EVSE YKLM S CG · - -- _ _ D W C Q _ _ ·· 0K W W V TO G v0 G E P G P N T L C 5 WmK I E K A G V L (J - - - Y K F R F e P - - 5 V C - os C T TL C S 0

Fig . I . Comparison of sequences of inhibitors in the Kunitz Soybean Trypsin Inhibitor Family.KSTI-Tia , Kunitz soybean trypsin inhibitorTi, (Kim et al. , 1985);WBTI-I , winged bean trypsininhibitor I (Yamamato et al., 1983);WBCI-3, winged bean chymotrypsin inhib itor 3 (Shibata eta\., 1988); ELTI-3, Erythrina latissima trypsin inhibitor 3 (Joubert et a\., 1985); PJTI-6,Prosop-sis julijiora trypsin inhibitor 6 (Negreiros et a\., 1991); BASI, barley a -amylase/subtilisininhibitor (Svendsen et a\. 1986); TCTI , Theobroma cacao trypsin inhibitor (Tai et aI., 1991).In each figure amino acid residues that are identical among the sequences are enclosed. Gapsintroduced to maximize alignment are indicated as -. The < Q at the amino-termininus ofPJTI-6 indicates a pyroglutamic acid residue. The reactive site bond location is indicated byan arrow and RS.

may be active against either trypsin or chymotrypsin, or both. In one case,Albizzia julibrissia (Odani et al., 1979) an a-chymotrypsin/elastase inhibitoris present, while Canavalia lineata has two Kunitz family inhibitors of sub-tilisin (Terada et aI., I994b,c) and one active against trypsin (Terada et aI.,1994a). Curiously, the two subtilisin inhibitors have the reactive site bondArg68-Gly69, hut are inactive against trypsin. All these inhibitors are highlyhomologous to KSTI, with approximately 160 to 200 amino acid residues,four of which are half-cystine residues (see Figure 1).The presence of multiple Kunitz type isoinhibitor forms in a single

plant species is common, e.g. in both the winged bean (Psopho carpustetragonolobus) and Erythrina corallodendron there are at least 8 isoin-hibitors (Yamamoto et a!., 1983, Joubert and Sharon, 1985). Many of theseinhibitors are composed of a single polypeptide chain like KSTI. However,the inhibitors in several genera, notably Albizzia (Odani et aI., 1979), Acacia

338 Karl A. Wilson

(Joubert, 1983; Lin et al., 1991), Adenanthera (Richardson et al., 1986) andProsopsis (Negreiros et al., 1991) consist of two disulfide-linked polypeptidechains corresponding to the amino- and carboxyl-terminal regions of KSTI.Presumably these are synthesized as a single-chained precursor that is thenprocessed to the mature two chain-form.Norioka et al. (1988) have shown that the KSTI family of inhibitors are

commonly found in members of the two more morphologically primitivelegume suborders Mimosaceae and Caesalpinieae, while the more advancedFabaceae have primarily the Bowman-Birk type inhibitors. In recent years ithas also become apparent that inhibitors of the KSTI family are not restrictedto the seeds of the Leguminosae . The best characterized of the non-legumeKSTI type inhibitors are the bifunctional subtilisin/a-amylase inhibitors inthe seeds of the Gramineae such as wheat (Triticum sp.) (Maeda, 1986; Mundyet al., 1984), barley (Hordeum vulgare) (Hejgaard et al., 1983; Svendsen etal., 1986), rye (Secale cereale) (Mosolov et al., 1986), and rice (Oryza sativa)(Ohtsubo and Richardson, 1992). These inhibitors are similar in size andsequence to KSTI. They inhibit the microbial serine proteinase subtilisin aswell as the endogenous cereal a-amylases (but not the exogenous a-amylasessuch as those of mammalian or insect origin) . The inhibition of subtilisin anda-amylase occurs at two distinct and independent reactive sites (Nesterenkoet al., 1987; Gvozdeva et al., 1994). In the inhibitor from wheat seeds, thereactive site for subtilisin appears to be Met34-Ala35, a site that does notcorrespond to the trypsin reactive site of KSTI. The a-amylase inhibitory siteappears to be localized in the carboxyl-terminal region, at or about Trp 161(Gvozdeva et al., 1994).Several other Kunitz family inhibitors have recently been described. A

21 kDa albumin in the cocoa seed (Theobroma cacao) has been shown tobe homologous to KSTI (Tai et al., 1991). The cocoa protein inhibits bovinetrypsin but not a-chymotrypsin, subtilisin Carlsberg, or barley malt a-amylase(Dodo et al., 1993). A trypsin inhibitor from white mustard seed (Sinapsisalba , Cruciferae) appears to belong to the Kunitz inhibitor family based onits molecular mass (18 kDa) and amino acid composition (142 residues with4 half-cystine residues) (Menegatti et al., 1985). Final confirmation of thisassignment must await elucidation of the sequence of this inhibitor. It shouldalso be noted that the KSTI protein family is found elsewhere in the plantother than just in the seed. Miracularin, the taste-modifying protein of theRichadella dulcificia berry pulp is homologous to KSTI, though apparentlynot a proteinase inhibitor (Theerasilp et al., 1989). Nodulin, a trypsin inhibitorexpressed in the senescent root nodules of the winged bean Psophocarpustetragonolobus, is also a homologue of KSTI, as is the product (apparentlynot a trypsin inhibitor) of a wound-responsive gene in hybrid poplar trees(Populus trichocarpa x P. deltoides) (Bradshaw et al., 1989). Homologues ofKSTI also appear to function as storage proteins (that lack obvious proteinaseinhibitory activity) in several plant systems, such as the sporamins of sweet


potatoes (Ipomoea batatas, Convolvulaceae) (Bradshaw et al., 1989) and aseed albumin (WBA-1) of winged bean tPsophocarpus tetragonolobus) seed(Kortt et al., 1989). Proteins of the KSTI family are thus widespread in plants,both monocots and dicots, and are presumabl y quite ancient in their origin .

C. The Bowman-Birk Inhibitor Family

Following the discovery and purification of KSTI by Kunitz, Bowman (1946)described the presence of a second trypsin inhibitor type in soybean (Glycinemax ). This inhibitor is distinct from KSTI in its solubility propertie s inmixed aqueous/organic solvents (such as aqueous ethanol and acetone). Thisinhibitory fraction was subsequently characterized by Birk and her colleagues(Birk et al., 1963 a,b; Birk , 1985). The inhibitor is thus now termed theBowman-Birk soybean tryp sin inhibitor (BBSTI) in recognition of theseworkers. Later work by Ikenaka's laboratory has elucidated the primary struc-ture of BBSTI (Odani and Ikenaka, 1972, 1973). Since the initial work ofBowman and Birk many representatives of the Bowman-Birk family of pro-teinase inhibitors have been described in other legume seeds (see Table 2).The presence of multiple isoinhibitor forms in the seeds of a particular plantspecies is common, and may be due to multiple gene product s, in vivo partialproteol ysis, proteolysis during purification, or a combination of these causes(see Section III above). The Bowman-Birk inhibitors appear to be localizedprimarily in protein bodies in the cotyledon cells of legumes (Horisbergerand Tacchini-Vonlanth en , 1983a).The Bowman-Birk inhibitors from legumes are single polypept ide chain

proteins of approximately 60 to 85 amino acid residues. They are notable inhaving 14 strongly conserved hal f-cystine residues in seven disulfide bonds(Odani and Ikenaka, 1973; Laskowski and Kato , 1980; Weder, 1981; Garcia-Olmedo et al., 1987). In general, a great degree of homology is observedbetween the Bowman-Birk type inhibitors of different legume species (Fig-ure 2). The Bowman-Birk inhibitors are double-headed , i.e. they generallycan simultaneously inhibit two molecules of proteinase(s) at the same time.The most common type inhibits both trypsin and chymotrypsin at the sametime (Weder, 1985; Shimokawa et al., 1984), such as is found in the clas-sical BBSTI (Birk, 1985), lima bean (Phaseolus lunatus) trypsin inhibitorIV (Krahn and Stevens, 1970; Tan and Stevens, 1971), and cowpea (Vignaunguiculata) (Mohry and Ventura, 1987). In this case trypsin is inhibited atthe first reactive site bond, typic ally Lys-Ser (e.g. Lys 16-Serl7 of BBSTI) ormore rarely Arg-S er (Shimokawa et al., 1983), while chymotrypsin is inhibit-ed at the second reactive site (e.g. Leu43-Ser44 of BBSTI , Phe-Ser or Tyr-Serin some other inhibitors (Wilson, 1981). Other reactive site combinations arepossible. Simultaneous inhibition of elastase and trypsin has been found ininhibitors from common bean (Phaseo lus vulgaris) (Wilson and Laskowski ,1975 ; Funk et al., 1993) and soybean (Glycine max) inhibitor ClI (Odani

340 Karl A. Wilson

TABLE 2

Bowman-Birk Family Proteinase Inhibitors of Legumes

Species Common Name Isoinhibitors References

Arachis hypogea Peanut 5 Norioka et al. (1982)

Canavalia lineata Jackbean sp. 2 Terada et al. (1994)

Cicer ariet inum Chickpea 6 Belew and Eaker (1976)

Glycine max Soybean 10 Tan-Wilson (1988)

Lathyru s sativ us Grass Pea 5 Roy (1972)

Lens culinaris Lentil 4 Mueller and Weder (1989)

Macrotyloma axillare 2 Joubert et al. (1979)

Phaseoluscoccineus Scarl et Runner Bean 5 Hory et al. (1976)

Pi lunatu s Lima Bean 6 Haynes and Feeney (1967)

P. vulgaris Common Bean , 3-6 Wil son and Laskowsk (1973)

Kidney Bean

Pisum sati vum Pea 6 Domoney et al. (1993)

Viciafaba Broad Bean 2 Warsy and Stein (1973)

Vigna angularis Adzuki Bean 5 Yokota et al. (1983)

V. radiata Mung Bean 4 Loren sen et al. (1981)

V. unguiculata Cowpea 2 Gennis and Cantor (1976)

and Ikenaka, 1977). Inhibitors that are double-headed for trypsin have beendescribed from mung bean (Vigna radiata) (Wilson and Chen, 1983) andpeanut (Arachis hypogea) (Norioka and Ikcnaka, 1983). In the mung bean thereactive site bonds are Lys26-Ser27 and Arg53-Ser54; in peanut inhibitor Allthey are Arg 19-Arg20 and Arg47 -Ser48. Intriguingly, the peanut inhibitoralso exhibits single-s ite inhibition of chymotrypsin, but not simultaneous-ly with trypsin inhibition. Apparently one of the Arg-X reactive sites alsointeracts with chymotrypsin. A similar observation has been made for thenon-canon ical interaction of the Arg-Ser reactive site of soybean inhibitorCII with chymotrypsin (Odani and Ikenaka, 1977).Examination of the amino acid sequences of the Bowman-Birk type pro-

teinase inhibitors reveals that the two reactive sites are located within tworegions of internal homology (Wilson, 1974; Weder, 1981; Odani and Ike-naka, 1982). These two domains are linked together by two segments ofthe polypeptide (i.e. in BBSTI, Ser25-Ser31 and VaI52-Phe57). The twodomains can be separated by cleavage of the two segments, e.g. with pepsinand cyanogen bromide (Odani and Ikenaka, 1978b; Townshend et al., 1982).These separated domain s retain inhibitory activity. Indeed, synthetic polypep-tides corresponding to parts of these inhibitory domains have been synthe-sized, e.g. corresponding to BBSTI residues 36-51 (Ando et aI., 1987) and


The Bowman-Birk Inhibitor FamilyI

. D D~- 5 5 K P C C _~10Q~A C T!5 N __ P ~OQ C R C 5~M R L ~O_ 5 C.5 0 H S S 5 DOE - S S K pee - 0 L C MeT~s M - - P P Q C H CAD I R L N - S C

,S DO S S 5 Y 0 DOE - Y 5 K pee - 0 L C MeT R S M - - P poe s rcra 0 I R L N - S C.Z P S Z - ssp p ee - B I e v e T A S I - - P P Q C r icrr B V R L B - S C

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. S G H H E H S T 0 Z P S Z - S S K P C (c - B H C A C) T K S ] - - P P Q eRe T 0 L R L 0 - 5 C. 1 IT E S K R Dec S V A C QeD K R L - - P poe GeM A R G F E - He

. M~R p w[El c c -ruN I K R L P T K PDP P W R .C NrnE L E P SOCR P W G 0 C C - 0 K A FTCl!!fKl.!:t N - ~ P PTe R .C MOE V K ~ - E C

. A T R P wm-c c - 0 R A I~F - - P MeR C MOM V E - ~ Q C

10

B8STtsa-errS8-0UG8-II 'MBTI -FLS -IVMeT!RDTl (1)RBTI (2)WGTI·II-4

BBSTIsa-er rS8-0 1IGS - II 'MBTI~F

LBTl-IVMCT1

RDTl (1 )RaT! ( 2)

WGTI-II-4

Fig . 2. Representative inhibitors of the Bowman-Birk Inhibitor Family. BBST!, the Bow-man-Birk soybean trypsin inhibitor (Odani and Ikenaka, 1972); SB-CII and SB-OIl , soybeanBowman-Birk type inhibitors Cll and DIl, respectively (Odani and lkenaka , 1977, 1978);GB-Il', garden bean inhibitor II' (Wilson and Laskowski, 1975); MBTI-F, mung bean trypsininhibitor F (Wilson and Chen, 1983); LB-IV, lima bean inhibitor IV (Tan and Stevens, 1971);MCTI , Momordica charantia trypsin inhibitor (Feng et al., 1989; RBTI (1) and (2), first andsecond halves of rice bran trypsin inhib itor (Tashiro et a\., 1987); WGTI-II-4 , wheat germtrypsin inhibitor 11-4 (Odani et a\., 1986). The two reactive sites (I and II) are indicated byarrows .

shown to be potent proteinase inhibitors. Studies examining the structure ofBBSTl in solution using two-dimensional proton NMR (Werner and Wem-mer, 1991, 1992) and of the Bowman-Birk type inhibitors from adzuki beanand peanut by x-ray crystallography (Tsunogae et al., 1986; Suzuki et al.,1987; Bode and Huber, 1992), clearly demonstrate the symmetric characterof the inhibitor molecule.We now know that the Bowman-Birk family of proteinase inhibitors is

not restricted to the Leguminosae. The best characterized non-leguminousBowman-Birk type inhibitors have been described from the cereal(Gramineae) grains, where they appear to be localized in the embryo and pos-sibly aleurone portion of the endosperm.This is in contrast to other inhibitors,such as the trypsin/a-amylase inhibitors of the CM-protein/napin inhibitorfamily (see Section II.G), which are found in the endosperm.Bowman-Birk type inhibitors have now been characterized from rice

(Oryza sativa) (Tashiro and Maki , 1978; Maki et al., 1980; Tashiro et al.,1987), wheat (Triticum spp.) (Odani et al., 1986), barley (Hordeum distichum)(Nagasue et al., 1988), Jobs' tears (Coix lachryma-jobi) (Ary et al., 1988),and foxtail millet (Setaria italica) (Toshiro et al., 1990,1991). Amino acidsequence analysis has shown that two types of Bowman-Birk inhibitors are

342 Karl A. Wilson

present. One form with approximately 65 residues resembles the legumeinhibitors in having two domains (e.g. wheat germ trypsin inhibitor 11-4,Odani et aI., 1986; Jobs' tears tryp sin inhibitor, Ary et aI., 1988; and thefoxtail millet trypsin inhibitors II and III , Tashiro et aI., 1990, 1991), yet issingled-headed for trypsin inhibit ion. The second form , with approximately130 amino acid residues, inhibits two mole cule s of trypsin per inhibitor mole-cule , and represents a tandem repeat of the basic two domain structure, i.e.four domains where domains I and III correspond to the first, amino-terminaldomain of BBSTI , while domains II and IV correspond to the carboxyl-terminal domain of BBSTI. In the cereal Bowman-Birk inhibitors the secondand fourth domain s appear to lack inhibitory activity (Tashiro et aI., 1987;Ary et aI., 1988).One Bowman -Birk type inhibitor, MCI-l, has been described from a mem-

ber of the Cucurbitaceae, the bitter melon Momo rdica charantia (Feng et aI.,1989). It has 77 amino acid residues and has a greater similarity to the dicotlegume Bowman-Birk inhibitors than to the monocot cereal inhibitors. MCI-Iinhibits two molecules of trypsin per inhibitor molecule, consistent with itstwo reactive site bonds, Lys18-Arg 19 and Arg45-His46 .

D. The Squash Trypsin Inhib itor Family

The smallest well characterized plant proteinase inhibitors have been found inthe seeds of the Cucurbitaceae (Polanowski et aI., 1980; Hojima et aI., 1982;Otlewski, 1990; Otlewski et aI., 1990). These inhibitors are polypeptides of29-32 amino acid residues, six of which are half-cystine residue s in threedisulfide bonds. Inhibitors of the squash trypsin inhibitor famil y have nowbeen described from a variety of cucurbits, including winter squash (Cucur-bita maxima) (Wilusz et aI., 1983), summer squash tCu curbita pepo) (Wiec-zorek et aI., 1985), cucumber tCucumis sativus) (Wieczorek et aI., 1985),watermelon (Citrullus vulgaris) (Otlewski et aI., 1987), red bryony (Bryoniadioica ) (Otlewski et aI., 1987), bitter gourd (Mom ordica charantia ) (Hara etaI., 1989) ,Momordica repens (Joubert, 1984), Ecballium elaterium (Favel etaI., 1989) , luffa gourd (Luffa cylindrica ) (Hatakeyama et aI., 1991) , and whitebottle gourd (Lagenaria leucantha) (Matsuo et aI., 1992). The presence ofisoinhibitors is common, and is often attributable to multiple genes or alleles .In part these isoinhibitors appear to be due to partial proteolysis . For example,in Cucurbita maxima CMTI-III is identical to CMTI-IV with the loss of threeamino-terminal residues (Otlewski, 1990) (Figure 3). A similar isoinhibitorpair is also found in C. pepo (Wieczorek et aI., 1985).Most of these inhibitors strongly inhibit bovine trypsin ( Kassoc values of

1010 to lOll M- I ) with a reactive site bond of Arg5-Ile6 or Lys5-Ile6 (num-bering of Cucurbita maxima CMTI-I) (Wieczorek et aI., 1985). In addition,a number of other serine proteina ses are inhibited to varying degrees, includ-ing the human blood coagulation system proteinases factors IXa , Xa , and


The Squash Proteinase Inhibitor Family

CMTI- ICMTI-IIICMTI- IVMRTI -lMCTI- IIMCEI -ICST I - lIbCPTI - IICVTI- IEETI - II

Fig. 3. The Squash Proteinase Inhibitor Family. CMTI-I, -III, and -IV, Cucurbita maxima(Otlewski, 1990); MRTI- I , Momordica repens (Joubert, 1984); MCTI-II and MCEI-I, Mor-mordica charantia trypsin inhibitor II and elastase inhibitor I (Hara et al., 1989); CSTI-lIb,Cucumis sativus (Wieczorek et al., 1985); CPTI-II, Cucurbita pepo (Wieczorek et al., 1985);CVTI-I , Citrullus vulgari s (Otlewski et al., 1987); EETI-II, Echallium elaterium (Favel et al.,1989). The arrow indicates the location of the reactive site.

XIIa (Hojima et aI., 1982; Wynn and Laskowski, 1990; Hayashi et aI., 1994),human plasmin (Otlewski, 1990; Otlewski et a\., 1990), human cathepsin G(Otlewski et a\., 1990), and plasma kallikrein (Hayashi et a\., 1994). Oneinhibitor from Momordica charantia, MCEI-I, has the reactive site bondLeu5-Ile6 and inhibits porcine elastase (Kassoc = 3 x 106 M- I) (Hara et a\.,1989).The small size of the squash family proteinase inhibitors and their potential

use as therapeutic agents, e.g. as anti-inflammatory agents (McWherter eta\., 1989) has made them the subject of several chemical synthesis studies.Wilusz et al. (1983) synthesized the 29 residue Cucurbita maxima CMTI-III inhibitor using solid phase synthesis methods, disulfide formation in thepresence of oxidized and reduced glutathione, and affinity chromatography onanhydrotrysin-Separose 4B. The resulting inhibitor was shown to be identicalto the natural inhibitor in its properties. In subsequent studies they producedreactive site mutants of CMTI-III using the same techniques. Substitution ofVal for Arg at position 5 (i.e. PI of the reactive site) increased the Kassoc of theinhibitor for human leuckocyte elastase and porcine pancreatic elastase byfactors of 107 and 103, respectively (Rolka et aI., 1991). Substitution of Pheat this site converted the polypeptide into a moderately strong chymotrypsininhibitor (Kassoc = 106 M- 1) . A more extensive series of substitutions wasproduced by McWherter et al. (1989). Inhibition of trypsin was observedwith a range of amino acids at the PI position of the reactive site bond,including Arg (the natural form of CMTI-III), as well as Leu, Phe, Met,and Ala. Chymotrypsin and cathepsin G were also inhibited by the analogswith reactive site Leu, Phe, Met, and Ala at the PI position, while humanleukocyte elastase was inhibited selectively by inhibitors with Val, Ile, andGly PI residues.

344 Karl A. Wilson

The Mustard Trypsin Inhibitor Family

MTI -2RTIATTI

MTI - 2RT IATTl

•1 10 20 3 0 4 0

~s EeL KEY G G 0 V G F P F CAP R I Fill T 'illY T R[]R E N K G A K Grn R

D SECLKEYGGDVGFGFCAPRlYPSFCVQRCRADKGALSGKo R K C L KEY G G 0 V G F S Y C A P R I F P T F C 0 Q NCR K N K G A K G G V

41 50 60

~'~G E G T N - - V K C LCD ymN 0 S P F 0 Q.

C 1 W G Q G S N - - V K C L C N F C R H G P .C R WEE N N A I G V K C L C N F C SEE P S 0 Q T L SRI.

Fig. 4. The Mustard Trypsin Inhibitor Family. MTI-2 , mustard trypsin inhibitor (Sinapsisalba) (Menegatti et al., 1992); RTI, rapeseed tryps in inhibitor tBrass ica napus) (Ceciliani etaI., 1994); Arabidopsis thaliana trypsin inhibitor (presumed) (GeneBank/EMBL AccessionNo. Z47386, Raynal, M., Grellet, E, Laudie , M., Meyer, Y., Cooke , R., and Delseny, M.). Thelocation of the reactive site is indicated by the arrow.

E. The Mustard Trypsin Inhibitor Family

Recently several trypsin inhibitors have been described from the seeds ofmembers of the mustard family, Cruciferae, that are homologous to each otherand lack any apparent relationship to other protein families. Inhibitors in thisfamily have been characterized and sequenced from white mustard (Sinapsisalba) (Svendsen et al., 1994) and rape seeds (Brassica napus) (Ceciliani etal., 1994) (Figure 4). In addition , the mRNAs for two putative inhibitors inArabidopsis thaliana have been cloned as cDNAs and sequenced (EMBLaccession numbers 247386 and 246816). The proteins themselves have notbeen isolated.The inhibitors consist of single polypeptide chains of approximately 60

amino acid residues with four intra-chain disulfide bonds. Bovine ,B-trypsinis strongly inhibited , while bovine a-chymotrypsin is also inhibited, thoughless strongly (Kassoc of 5 x 109M- 1 and 2 x 106M- 1, respectively). The reac-tive site bond has tentatively been identified as Arg20-Ile2l in the Brassicainhibitor based upon the cleavage of this bond during affinity chromatographyon trypsin-Sepharose (Ceciliani et aI., 1994).

F. The Potato Proteinase Inhibitor I Family

The potato proteinase inhibitor I (PPI-I) family is named after the inhibitorfirst isolated from potato tubers (Melville and Ryan, 1972; Richardson, 1974;Cleveland et al., 1987). PPI-I itself is an oligomeric protein of approximately41,000 molecular weight, made up of 8,100 molecular weight protomers.Each protomer is a chain of 86 amino acid residues, including two half-cystine residues. PPI-I inhibits chymotrypsin strongly at a single site perprotomer. Trypsin is also inhibited weakly. Members of the PPI-I family aswell as the potato proteinase inhibitor II family in the Solanaceae have beenshown to greatly increase in concentration in the aerial tissue in response to


wounding (Ryan, 1979 ,1990), as well as being expressed transiently in thefruit (Pearce et aI., 1988). This is consistent with these inhibitors servingas components of a defensive system aga inst herbivores (Ryan, 1990). Noreports have appeared indicating the presence of either PPI-I or PPI-II inthe seeds of the Solanaceae. However, other studies have demon strated thatPPI-I homologues are present in a wide variety of plant families, includingthe Legum inosae, Cucurbi taceae, Amaranthaceae , Gramineae, and possiblythe Polygonaceae.In the legumes these inhibitors have most often been identified as subtil-

isin inhibitors. Such inhib itory activity has been identified in a wide vari-ety of legumes using either direct activity assays (Seidl et aI., 1988) or byinhibitor activity stainin g after electrophoresis or isoelec tric focusing (Loren-zo et aI., 1989; Kapur et aI., 1989). Subt ilisin inhib itors of the PPI-I familyhave been purified and charac terized to varying extents from Canaval ia ensi-f ormis (Lorenzo et aI., 1989), Dolichos bifiorus (Bodhe, 1991), Phaseolusvulgaris (Seidl et aI., 1982; Mosolov et aI., 1983), Vicia faba (Svendsen etaI., 1984), Vigna angula ris (Yoshikawa et aI., 1985), Vigna radiata (Kapur etaI., 1989), and Vigna unguiculata (Vartak et aI., 1980). Multiple isoinhibitorforms are found in most spec ies. These proteins are approximately 9,000 to13,000 molecular weight, and they are notable for the absence of half-cystineresidues (with the exception of one P. vulgaris subtilisin inhibitor which isreported to have four half-cystines (Seidl et aI., 1982)). Sequences of the Viciafa ba and the Vigna angularis inhibitors demonstrate that these inhibitors arehighly homologous to PPI-I (Figu re 5). All strongly inhibit subtilisin (variousenzyme forms) with reported Kassoc values on the order of 5 x 109 M- 1

(Yoshikawa et aI., 1985; Bodhe, 1991). Mammalian pancreatic trypsin andchymotrypsin are not inhibited , although pancreatic elastase or human leuko-cyte elastase may be weakly inhibited by some inhibitor species (Seidl etaI., 1982; Svend sen et aI., 1984). Inhibition of various microbial alkalineproteases has also been described , as with the Vicia faba and Canavalia ensi-formis inhibitors. Inhibition of cys teine proteases such as papa in, ficin, orchymopapain has not been reported .A number of other representatives of the PPI-I family have been identified

in the dicots (Figure 5). Such inhibitors have been studied in Momordicacharantia (Cucurbitaceae), where a trypsin inhibitor with no disulfide bonds(Zeng et aI., 1988) and an inhibitor of acidic amino acid-specific Strepto-myces griseus endopeptidase with one disulfide bond (Ogata et aI., 1991)have been found. An inhibitor of trypsin and Hageman factor (Factor XIIa)from Cucurbita maxima has also been sequenced (Krishnamoorthi et aI.,1990), suggesting that inhibitors of the PPI-I family may be widespread inthe cucurbits. Inhibitors of the PPI-I family have also been described in theAmaranthaceae, i.e. in Amaranthus caudatus (Hejgaard et aI., 1994) andA. hypochondriacus (Valdez -Rodriguez et aI., 1993). Both strongly inhibittryps in and chymotrypsin, while the A. caudatus protein also inhibits subtil-

346 Karl A. Wilson

The Potato Proteinase Inhibitor I Family

10 '20 ) 1) 40

.Q E Q G T N P S Q EON V P L P R N Y K 0 ALE TNT P T[!)T S W PEL V G V T A E 0 A E T K~K _• . . R T S W PEL V G v S A E E A R · K I K -

. 1'1. R E C P G~O E W PEL V G E Y G Y K A A ' A I I E R. Ae -S Q C Q G K R S W P'LV G S T G A A A K A V I E R

. S S C PO K SS W P H L V G V G G S V A K A I I E R. Y PEP T E G S I 0 A S G A K T S W P E V V G M S A E K A K E I I L ~

( Z,V, S,SIK K PEG V N T GAG 0 R H N L K T E W P s L V G K S VE E A K K V I L •

. S FEe 0 0 K L Q W PEL I G V P T K L A K E I I E K

VAS I - I

VFSIAHTIBOlACMTI -VHVCI - I C

HVCI- 2PPI-I

Fig. 5. Representative members of the Potato Proteinase Inhibitor I Family. VASI-I,Vigna angularis subtilisin inhibitor (Nozawa et al., 1989); VFSI, Vicia faha subtilisininhibitor (Svendsen et aI., 1984); AHTI , Amaranthus hypochondriacus trypsin inhibitor(Valdez-Rodriguez et aI., 1993);BGIA,Momordica charantia (bitter gourd) inhibitor for acidicamino acid specific endopeptidase (Ogata et aI., 1991); CMTI- V, Cucurhita maxima trypsininhibitor V (Krinamoorthi et al., 1990); HVCI-IC and -2, barley CI-1 and CI-2 inhibitors(Svendsen et al., 1982); PPJ-I, potato protease inhibitor I (Richardson , 1974). The position ofthe reactive site bond is indicated by the arrow.

isin. The inhibitors from both species are 69 amino acid residues, and differfrom each other in only two amino acid residues. It seems likely that the'permanent' trypsin inhibitors in buckwheat seeds (Fagopyrum esculentum) ,with 51 to 67 amino acid residues (two of which are half-cystine), are alsomembers of the PPI-I family (Kiyohara and Iwasaki , 1985).Members of the PPI-I family have also been demonstrated in monocots.

Two distinct chymotrypsin inhibitors, CI-l and CI-2 have been characterizedby Boisen et al. (1981). Both were found to consist of several isoinhibitorforms (i.e. CI-la and -1b; CI-2a, -2b, -2c, and -2d). They have apparent mole-cular weights of 22,000 and 12,000, respectively, on gel filtration at pH 4.9.However, amino acid analysis and sequence analysis (Svendsen et al., 1982;Jonassen and Svendsen , 1982) indicate that the inhibitor polypeptides are ofsimilar size (77 and 83 residues for CI-lc and CI-2 respectively), suggestingdifferent degrees of self-association. The sequences of CI-l and CI-2 arehomologous to each other and to PPI-I (Figure 5). It seems likely that similarinhibitors are found in other cereal grains. Mosolov et al. (1984) partiallycharacterized a subtilisin/chymotrypsin inhibitor from com (Zea mays) seedsthat is very similar in molecular weight and amino acid composition.The inhibitors of the PPI-I family appear to follow the canonical reactive

site model for inhibitor/protease interactions. The Vigna angularis subtilisininhibitorASI-II reactive site has been identified as Ala49-Asp59 by incubation


with catalytic amounts of subtilisin BPN' at pH 3.8 (Nozawa et al., 1992).The two chains of the modified inhibitor are held together only by non-covalent interactions (due to the lack of disulfide bonds in the molecule) andare readily separated by reversed-phase HPLC. Neither the 49 residue amino-terminal peptide nor the 24 residue carboxyl-terminal fragment are activeas subtilisin inhibitors. However, an equimolar mixture of both fragments isactive. Reaction of the reconstituted modified inhibitor with subtilisin at pH7.6 results in the production of the native inhibitor, further supporting theapplication of the reactive site model of Laskow ski and Sealock (1971).The molecular structure of barley CI-2 has been determined by X-ray

crystallography both as the inhibitor alone and in complex with subtilisinNovo (McPhalen et aI., 1985; McPhalen and James, 1987). Comparison ofthe free and complexed inhibitor structures indicates that the reactive site loophas considerable flexibility. This may contribute to the ability of the proteinto inhibit a range of proteolytic enzymes (McPhalen and James, 1987).In concluding this section, it should be noted that the PPI-l family of

inhibitors is of special interest from an evolutionary point of view. Not onlyare members of the family found in plants, as discussed above, but also inan animal, as represented by eglin, an elastase/cathepsin G inhibitor from theleech Hirudo medicinalis (Svend sen et aI., 1982).

G. The CM-Protein/Napin Trypsin Inhibitor Family

This inhibitor family was originally called the cereal trypsin/a-amylaseinhibitor family . However, in light of the recent description ofmembers of thisfamily in dicots (see below), a broader descriptive title, the CM-protein/napintrypsin inhibitor family, will be used here. This is a complex family of pro-teins containing both inhibitory and non-inhibitory proteins, in both monocotsand dicots. The relationship of many of the members to this family has onlybecome apparent in recent years as the accumulation of amino acid sequencedata has made comparisons for homology possible.A trypsin inhibitor from barley (BTl), molecular mass 14 kDa, was first

described by Mikola and Suolinna (1969) , and subsequently sequenced byOdani et aI. (1983a). BTl forms a 1:1 complex with bovine trypsin, but isinactive against chymotrypsin, pepsin, microbial serine proteinases, and theendogenous barley proteinase. Odani et aI. (1983a) noted that the sequence ofBTl is homologous to that of a inhibitor of exogenous a-amylases from wheat,Clll (Kaslan and Richardson, 1981). It is now known that this homologyextends to a whole family of cereal grain endosperm proteins known asthe CM-proteins due to their solubility in chloroform/methanol mixtures(Rodriguez-Loperena et aI., 1975; Salcedo et aI., 1978, 1982).The members of this protein family exhibit a wide range of activities : as

trypsin inhibitors, as a-amylase inhibitors, as bifunctional trypsin/a-amylaseinhibitor, or as (apparently) non-inhibitory storage proteins. All members of

348 Karl A. Wilson

The CM Protein/Nepin Inhibitor Femdly

MTFI

fMATlBTlRT!WAIRCSP

BNSPBNTI

MTFIFHATIBTlRTlWA !

RCSFBNSPBNTI

MTf'I

FMATIBT lRTIWAI

RCSPBNSPBNT I

.:ng~~;~n ~ ~ ~ ~ ~ ~~d (~~ ~ ~g~ ~ n~~ ;~~~n~ ~ ~ ~. ~ ~ ~ {~. 5 G P W 5 w e N P A T G Y K V 5 A L T G e R A H V K L Q - ~- V G 5 Q v P E • - - - - - - A V L. F S 0 Q G C R - GO 1 0 E 0 0 N L ROC 0 E Y I K 0 0 v S G O G P R R . .Q E R 5

. 5 A. G P F R IP K C R - K E F 0 Q A Q H L R Ae Q Q W H H K Q A M Q S GO G P S • . P 0 G P O 0 R P P L

. P A G P F R IP K C R - K E: F 00 11. Q H L R Ae Q Q W L H K 0 A M Q 5 G G G P S . • P 0 G PO 0 R P P L

41 50 60 70 BO

K R R R E~A 0 I P A Y -~R C AL S I L M 0 G A I P P G P O A Q LE G R L - E 0 LPG - - -KAR CCRQL E A IP AY- C R C E AV R I L M DGV V TPS GQ - -HEGR L LQD LPG---KR RCC D EL S A I P AY-CR C E A LR I I MQGVVT WQG A--FE G A YFKDS PN - --K R R C CDEL L AIPA Y - CR CEA L RI LM D G V VT Q Q G V-- F E G G Y LK D MPN - --R R DC C Q Q L A D I N N E W C R C G O L S S M L R A V Y Q Q L G V - - R E G - - K E V L P G - - -LR GC CD H LKQMQSQ- C R CEGLRQA - -- -- - -IQQO ·O LQG - -O N V FEA.FRL Q Q C C N E L H O E E P L- C V C P T L K G A S K A V K O O I O O O O Q Q Q G K Q Q V P S R I Y QL O Q C C N E L H O E E P L~ v PT L KGAA KAVK Q QQ Q O Q Q O O OM · O Q VPSRI YQ

90 10 0 110

~ - • - ~p REV Q R G F A A T L V TEA E~N L A TIS .- - - - C P R Q v Q R A F A P K L V T EVE C N LA T I H G G P FOOL S LL G A G E .~ • - - C P R E R Q T S Y A A N L V T POE C N L G T I H GSA yep E L Q P G Y G.~ • • - C P R V T Q R 5 Y A A T L V A P Q E C N L P T I H G S PVC P T L Q A G Y .•• - - C R K E V M K L T A A S V P E • ~ v C K V PIP N P SG D RAG V@]V G D w e A V PO V.

T A A N L P S "fiN I Q 0 V S - - - - - - 0 C R F .T AT HLP K V C N I Q 0 v S - - - - - - v C P F Q K T M P G P S Y .T AT H L P K V C - • - - - - • • - - • - V C P KKK T M P G P S .

Fig. 6. Members of the CM Protein/Napin Inhibitor Family. MFfI, maize trypsin/Hagemanfactor inhbitor (Mahoney et al., 1984); FMATI, finger millet a-amylase/trypsin inhibitor (Cam-pos and Richardson, 1983); BTl , barley trypsin inhibitor (Odani et al., 1983); RTl, rye trypsininhibitor (Lyons et al., 1987); WAI, wheat a -amylase inhibitor 0.28 (Kaslan and Richard son,1981); RCS?, castor bean storage protein (Sharief and Li, 1982); BNSP, Brassica napus NapinA (Josefsson et al., 1987); BNTI,Brassica nap liS rapifer (kohlrabi) trypsin inhibit or (Svendsenet al., 1989). The half-cystine residues and residues common to all proteins are enclosed.

the cereal grain CM-protein group are similar in size (a single polypeptidechain 112-146 amino acid residues long) with 10 half-cystine residues in 5disulfide bonds. These seed proteins are thus very rich in sulfur-containingamino acids, and may fonn a significant depot for this element in the seed.Trypsin inhibitors of this type have now been isolated from com (Zea mays)(Swartz et a\., 1977; Mahoney et a\., 1984; Corfman and Reeck, 1982),teosinte (Zea mexicana, the presumed progenitor of com) (Corfman andReeck, 1982), and rye (Secale cereale) (Lyons et a\. , 1987). A bifunctionaltrypsin/a-amylase inhibitor is found in ragi, the Indian finger millet (Eleusinecoracana) (Campos and Richardson, 1983). All are homologous to other CM-proteins such as the wheat a -amylase inhibitors (Shewry et a\. , 1984, Barberet a\., 1986, Garcia-Olmedo et a\. , 1987, Halford et a\., 1988) (Figure 6).Members of this protein family are also found in the seeds of dicots.

Odani and coworkers (1983) first noted that BTl was homologous to the2S storage protein of castor bean (Ricinus communis) . The latter consists oftwo polypeptide s of 34 and 61 amino acid residues each, and is a memberof a group of 2S storage proteins typified by the napins of the Brassica(Josefsson et a\., 1987), the conglutin 62 of lupin (Lupinus angustifoliusi


(Lilley and Ingli s, 1986) and Brazil nut 2S sulfur-rich protein (Bertholletiaexcelsa) (Ampe et al., 1986).Two trypsin inhibitors belonging to this family have now been described

from dicot seeds, from the charlock (Sinapsis arvensis) and kohlrabi (Brassicanap us rapijera ) seeds (Sve ndse n et al., 1994; Svendsen et aI., 1989). Theyare approx imately 13 kDa to 15 kDa in size, and consist of two polypeptidesof 39 and 85 to 87 amino acid residues, bound together by one or moredisul fide bonds. In all, the molecule has four disulfide bonds. Like napin,the two-chained inhibitors are presumably initially synthes ized as a single-chai ned precursor that is processed to the mature protein of two chains. Boththe Sinaps is and Brassica protein s inhibit bovine trypsin, and , to a lesserex tent, subtilisin DY. The Sinaps is protein also inhibit s chymotrypsin, whilethe Brassica inhibitor is inacti ve toward chymotrypsin. Both plant speciescontain at least two isoinhibitor form s of this family of trypsin inhibitors.While the napin-like trypsin inhibitors from the crucifers Sinapsis and

Brassica and the related 2S dicot seed proteins are clearly homologous to thecereal seed Clvl-protein s, there are distinct differences. As noted above, themonocot proteins con sist of a single polypeptide chain with 5 disulfide bonds.In contras t, the dicot proteins in their mature forms have two polypeptidechains with a total of 4 disulfide bonds.

H. The Protein Z/Serpin Family

The serpins (ser ine protein ase inhibitors) (Protempa et aI., 1994) are a familyof proteinase inhibitors that are common constituen ts in animal blood plasmaand other fluids . They have been found in animals ranging taxonom icallyfrom primates to insects and Schistosoma. Surpri singly, both inhibitory andnon-inhibitory homologues of the animal serpins have been found in theendosperm of cereal gra ins. Hejgaard et aI. (1985) noted sequence homologyof the major barley endos perm albumin, protein Z, with a I-antitrypsin andother members of the serpin family (Figure 7). However, no inhibitory activityaga inst trypsin , chymotrypsin , pancreatic elastase, subtilisin, or Aspergillusprot ease (Hejgaard et aI., 1985) was found with barley protein Z. Recentl y,Lundgard and Svensson (1989) have described a minor protein Z-like 39 kDaprotein in barley which is an inhibitor of a-chymotrypsin but has little orno effec t on tryp sin , subtilisin and several other microb ial proteina ses , andtwo malt cysteine endopeptidases. Rosenkrands et aI. (1994) have recentlyisolated a protein Z-like chymotrypsin inhibitor from wheat grain by affinitychromatography on chyrnotrypsin-aga rose. It also exhibits a high degree ofhomology with the serpins in the region sequenced.

350 Karl A. Wilson

The Protein ZjSerpin Family

BZ3 9WSZCIBZBSZ4a l·P IPAl -2

Fig. 7. The Protein Z I Serpin Family - alignment of partial amino acid sequences . BZ39,barley protein Z-like 39 kD protein (Lundgard et al., 1989); WSZCI, wheat seed protein Z-Iikechymotrypsin inhibitor (Rosenkrands et aI., 1994); BZ, barley protein Z (Hejgaard et al. , 1985);BSZ4, barley protein Z4 (Brandt et al., 1990); a I-PI , human a I-proteinase inhibitor (Kurachiet al., 1982); PAI-2, human plasminogen activator inhibit or (Huber and Carrell, 1989).

I. The Maize BifunctionallnhibitorlThaumatin Family

Richardson and coworkers (1987) have described a bifunctional protein inthe seeds of com (Zea mays) that is an inhibitor of both bovine trypsin andthe a -amylase of the beetle Tribolium castaneum. This inhibitor cannot beassigned to any of the homology families described above. It consists of asingle polypeptide chain of 206 amino acid residue s with 16 half-cystineresidue s. This inhibitor appears to be identi cal to zeamatin, an antifungalprotein isolated from com seeds by Roberts and Selitrennikoff (1990; seealso SWISS-PROT protein sequence database accession P33679), on the basisof molecular weight and the amino-terminal sequence. Zeamatin is detectedby its ability to inhibit the growth of Candida albicans, Neurospora crassa,and Trichoderma reesei, apparently by permeabilizing the fungal plasmamembrane. Unfortunately, zeamatin has not been analyzed for protease oramylase inhibitory activity.No other proteinase inhibitor homologous to the com bifunctional inhibitor

has been identified in seeds. However, it has become apparent that the inhibitoris a member of a larger plant protein family. This family of homologues(Figure 8) includes the sweet tasting protein thaumatin (from the fruit of theshrub Thaumatococcus danielli, Iyengar et aI., 1979), a protein induced bylow water potential, osmotin (Singh et aI., 1989), the pathogenesis-relatedprotein from tobacco (Cornelissen et aI., 1986) , the P21 protein of stressedsoybean leaves (Graham et aI., 1992), and similar proteins from a varietyof plant sources. There have been no publi shed reports of inhibitory activityresiding in any of the other proteins. All of the other members of this proteinfamily (with the exception of thaumatin) appear to represent proteins whosesynthesis is induced in response to environmental or biological stress on theplant. The bifunctional inhibitor of com may also exhibit such behavior duringseed development.


The M. ize Bifunctional Inhibitor!Tbaum.atin Pamily

MTAtTHAOOSMTS BP2 1

MTAIT H A.U

OSMT5 8 P2 1

MTAlTHAOOSMTSB P21

MTAtTH AUOSMTS8P2 1

OSMT

'0 '0 rn0 ~ ~Oilly FTVvmorn· • TV "AASV• v- - - - - G~G "om""G•sm" ,TA. AGTTAA" 'rnA"TGA TFE IVNRC SY TVWAAASIC OD AA L D AOG ROLN SOIlS W T IN VEP O TNGOK IH' AR TD

A T IE VRNN CP YTVWA A ST P I -· · p -OQ OR R LD R GOT NVI NA PRG T KMAR VWGR TNAR F E tT NRC TY T VW A A S V P V ·· - - -O Q O V Q L N P O Q S W SV DV P A O TltO A R V W A R T Q

~omO Asm"~·~rn.: TGDeGGv'~ °mTGYG" A~': TLA' YAm"0': " "LrnF•Dr .':"c DGF"C'f F D D S G S I C K T OD CG GLL R C KRF OR P P T T LA KF S LN QY-G K D YI D I SNIKGFH

C N F N A A G R l' C QT GD C Oc;V L QCT O H GK PPN TL AE YA LD QF SG LDFWD I S LLDG FNC N FD GSG R G C Q TG D C GG V LDCK A YGAPP N TL A B YG LNGPN N LDF F D ISL VD G PN

vmy .'~'"cmD GGS- - G rn.'~OG • "illAVDV"'~o"~.A' L" -c D'rn"v~" " Am. vm'~" n • YCCVVP MN F S P T T R - - -G CR -G V RC A A D IV O QC P AK LK A P GO G C N D ACTV F Q T S E Y CC _

I P M T F- P T N PS G G K C H --A L C T A - I N G E C P A E L R v p - o a C N N P C T T F G G O O Y C C _VP M D FS P T S N - -- G C T R G I S C T A D I NO Q C P S Ii: L K T O -O G C N N P C T V F K T D O Y C C N

G.':0A" "~H~T "/rn70"Y[]"GQ C• :~OV[]Y•"meA/:Orn F~C •AG-T:mOO"mvFCP- - T T 0 Ke G PTE Y S R P P K R L C PO A F S Y V L D K P T - T V T C P G S - S N Y R V T F C P T A .- -T Q R P C G P T F F S K F F K Q RC P O A Y S Y P - O D P T 5T F T C P G G S T N Y RV I F C P N G Q A H50 - -- 5 0 D Y R F OR Y y p K D PP5 FT CNGG - T D RVVF CP

P N F P L E M P G S 0 E V A K.

Fig. 8. The Maize Bifunctional Inhibitor/Th aurnatin Family. MTAI, maize trypsin/a -amylaseinhibitor (Richardson et al., 1987); THAU, thaumatin (Iyengar et aI., 1979); OSMT, osmotin(Singh et aI., 1989); SBP21, soybean leaf protein P21 (Graham et al., 1992). Residues commonto all four proteins are enclosed.

J. The Phytocystatin s

The majority of work on proteinase inhibitors has concentrated in inhibitorsof serine proteinases. Thi s is reflected in the considerable body of literature,reviewed above , that has acc umulated conc erning these inhibitors. Howev-er, as recognition of the importance of cysteine proteinases has increased,so has the interes t in natural inhibitors of these proteinases. Most studieshave utilized the relati vely inexpensive and easily obtainable papain, ficin,or bromelain. Some later studies have used mammalian enzymes such asca thepsin H (Kondo et al., 1990), the cysteine proteinases from the midgutof coleopteran pests on stored grains and pulses (Liang et al., 1991; Camposet al., 1989) , or endogenous plant seed/seedling cysteine proteinases (Abeet al., 1991; Baumgartner and Chri speels, 1976). These studies have led tothe characterization of a number of cysteine proteinase inhibitors from seeds.Thus far, all of the well characterized (i.e . sequenced) inhibitors belong tothe phytocystatin inhibitor family of the cystatin superfamily (Barrett et al.,1986).By far the best studied of the plant cystatins are the inhibitors from rice

(Oryza sativa) grains, the oryzacystatins. Rice contains two oryzacystatins , Iand II, which are each single polypeptide chain s of 102 and 107 amino acidres idues, respecti vely. Ne ither contains half-cystine (Abe et al., 1987a,b; Araiet al., 1991 ; Kondo et al., 1990). Oryzacystatins I and II exhibit 55% identityto each other (Figure 9), as well as sequence homology to the family 1 and2 anima l cystatins. The oryzacystatin s resemble a combination of charactersfrom these two families, lacking disulfide bonds as in family 1 cystatins (the

352 Karl A. Wilson

The Cystatin Family

MCI

QC·I

oc-rrCPCSBCNFC- )

CC

MCIQC . !

oc-r rCPC

SBCWFC· }

CC

" C Ioc- rOC· lICPC

CC

10 20 3 0 40 50.M R I( H R I V S L V A AL L V L L A L A A V SST R S T 0 K E S V A D NA G M L AG 0 - • 1 K 0 V - rnA - N

. M S S - - 0 G G P V L G G V E P Vffi".M A B Iii: A Q S H AR E G G R H PRO - - P A G R

.M A A L G G • - - - N R 0 V A G N. 0 P T D I T G A

. Y a N T G G Y T [!]V P D. S Iii: DR S R. L L G A po V P V V E HD B@] -

Fig. 9. The Cystatin Family of Inhibitors. MCI, maize cysteine proteina se inhibitor (Abe etal., 1992); OC-I and OC-II, oryzacystatins 1and II (Abe ct al., 1987; Kondo et aI., 1990); CPC,cowpea cystatin (Fernandes et aI., 1993); SBC, soybean cystatin (Brzin et al., 1990); WFC-3,Wisteria cystatin 3 (Hirashiki ct al., 1990); CC, chicken (egg) cystatin (Co lella et aI., 1989).

stefins), but exhibitin g more overall similarity to the amino acid sequences ofthe animal family 2 cystatins (Abe et aI., 1987a; Barrett et aI., 1986). Becau seof this, Abe et al. (l987a) proposed a new subfamily, the phytocystatins, toreflect this combination of characters. The cDNA of both oryzacys tatins havebeen cloned, and their expression during seed development examined (Abe etaI., 1987a; Kondo et aI., 1990). A papain inhibitor similar to the oryzacystatinshas been described in com (Zea mays) (Abe and Whitaker, 1988 ; Abe et aI.,1992).While oryzacystatins [ and n are very similar in structure, they differ

significantly in their interaction with papain and cathepsin H. Oryzacystatin Iinhibits papain more effectively than cathepsin H (K, of2 x 10- 8 M and 0.8 x10- 6 M, respectively), while oryzacystatin II is a better inhibitor of cathepsinH than papain (K, of 1 x 10- 8 M and 0.8 x 10- 6 M, respectively) (Kondoet aI., 1990). Three cysteine proteinases have been identifi ed in germinatingrice seed s, oryzains a and (3, which resemble papain in amino acid sequence,and oryzain "Y, which more clo sely resembles cathepsin H. Abe et aI. (1991)have suggested that oryzacystatin I may be targeted toward oryzains a and(3, while oryzacystatin n is directed toward oryzain "Y. In addition to theseplant and mammalian proteinases, oryzacystatin is a potent inhibitor of themidgut cysteine proteinases of the rice weevil Sitophilus oryzae , and the redflour beetle Tribolium castaneum, as well as proteinases from several otherstored-grain insect pests.Phytocystatins have also been found in the seeds of legumes, and have now

been characterized from soybeans (Glycine max) (Brzin et a!., 1990; Hines et


al., 1992), cowpeas (Vigna unguiculatai (Fernandes et aI., 1993), andWisteriafioribunda (Hirashiki et aI., 1990). They range in size from approximately16 to 12 kDa, and like the cereal phytocystatins lack half-cystine (Figure 9).All three inhibit papain, and, at least in the case of the cowpea and soybeaninhibitors, also interact with catalytically inactive carboxymethyl-papain (Fer-nandes et aI., 1991; Brzin et al., 1990). All are homologous to the cereal grainphytocystatins (Figure 9).It seems likely that plant seeds may contain a diversity of inhibitors active

against cysteine proteinases. A number of other inhibitors of cysteine pro-teinases have been described from legume seeds, but their relationship to thephytocystatin family cannot at this point be determined. Oliva et al. (1988)have described a high molecular mass papain inhibitor from Enterolobiumcontortisiliquum beans. It consists of a single 60 kDa polypeptide chain whichinhibits bromelain in addition to papain. The seeds of Wisteria also containa large (41 kDa) papain inhibitor as well as the 15 kDa phytocystatin notedabove (Hirayama et al. , 1989). Other low molecular mass cysteine proteinaseinhibitors have been described that remain problematical because of the lackof sufficient structural characterization. Zimacheva and Mosolov (1995) havenoted the presence of two 14 kDa inhibitors in soybeans which inhibit papain,ficin, and bromelain. However, unlike the soybean cystatin , these inhibitorsare irreversibly denatured in the presence of reducing agents such as 5 mmL-cysteine, which are usually included in assays utilizing papain.

K. Inhibitors ofMetallo- and Aspartic Proteinases

It should be apparent from the above survey that plants have developed awide array of inhibitors localized in the seed and directed toward serineand cysteine endopeptidases. Our knowledge of inhibitors of the other twomechanistic classes ofendopeptidases (the metalloproteinases and the asparticproteinases) is in contrast very limited. This may be due in part to a bias inresearch towards the serine and cysteine endopeptidases. However, it is alsopossible that there has been relatively little selective pressure for plants todevelop such inhibitors in the seed (see Section III).One apparent inhibitor of metalloproteinase has been described from the

buckwheat seed (Fagopyrum esculentum) (Voskoboinikova et aI., 1990).Thedegradation of the buckwheat 13S storage globulin is initiated upon imbi-bition by a 38.9 kDa metalloproteinase (Dunaevskii et aI., 1983). The pro-teinase and storage protein are present in the protein bodies ; also present inthese organelles is a 12 kDa inhibitor of the metalloproteinase. The in vitroinhibition of the proteinase by this inhibitor can be relieved by the additionof divalent metal ions such as Zn2+, mg2+, and Co2+. In addition, dialysisof the inhibitor against the same cations inactivates the inhibitor, prevent-ing the inhibition of the buckwheat metalloproteinase. This suggests that theinhibitor binds to the proteinase through the required metal ion, leading to

354 Karl A. Wilson

inhibition . Elpidina et al. (1991) have hypothesized that in the dry buckwheatseed the proteinase exists primarily or exclusively as a complex with theinhibitor. Upon imbibition, phytin, stored as globoids in the protein bodies,is hydrolyzed, releasing divalent metal cations. These in tum compete forbinding to the inhibitor with the proteinase, resulting in the release of freeproteinase and the initiation of storage protein degradation.An inhibitor of the mammalian metallo-carboxypeptidases A and B has

been isolated from the kidney bean (Phaseolus vulgaris) by Hojima et al.(1979). This inhibitor also appears to work by complexing to the metal ionnecessary for enzymatic activity. Its action is not limited to the carboxypep-tidases, as it can also inhibit the phosphatase from calf intestine. However,the very low molecular weight (< 0.5 kDa) of this inhibitor disqualifies it asa protein, in spite of its positive reaction with ninhydrin.No well characterized inhibitor of aspartic proteinases has yet been

described from seeds , although an inhibitor of the aspartic proteinase cathep-sin D has been isolated from potato tubers (Mares et al., 1989).

III. The in vivo Function of Seed Proteinase Inhibitors

The seeming ubiquity in seeds of protein inhibitors of serine proteinases (andpossibly also inhibitors of cysteine proteinases) suggests that these proteinsserve an important function in the seed. Several such functions have been pro-posed (Pusztai, 1972; Ryan, 1973, 1979; Richardson, 1977). One possibilityis that these inhibitors regulate endogenous proteinases in the seed, either dur-ing seed development or germination and early seedling growth. This wouldbe analogous to the situation in many animal systems (Fritz and Tschesche,1971). Alternatively, it has been suggested that the inhibitors serve to protectthe seed from fungi or other microbes, small invertebrates such as nematodes,or various insect pests that would otherwise attack the quiescent or germinat-ing seed. Finally, data exists supporting the idea of some proteinase inhibitorsacting as significant storage proteins. Each of these will be examined below.In fact, it seems likely that in some cases a proteinase inhibitor may actuallyserve several functions, either simultaneously or at different stages in theseed 's natural history.

A. Seed Proteinase Inhibitors as Regulators ofEndogenous Proteinases

The proteinase inhibitors in animals generally appear to function as regulatorsof proteolytic activity, e.g., in the activation of the blood clotting stream, inthe storage and secretion of pancreatic serine proproteinases, in neutrophilphagocytosis, etc. (Heimburger, 1974). It is therefore not unexpected that thismodel would be suggested for the plant proteinase inhibitors. However, onlya few cases have been reported where the seed proteinase / seed proteinase


inhibitor pair has been identified. Cysteine proteinase / inhibitor pairs havebeen found in rice (oryzain a / oryzacystatin I) (Abe et aI., 1991), in scotspine (Pinus sylvestris) (Salmia and Mikola, 1980; Salmia, 1980), and in themung bean (Vigna radiatai (Baumgartner and Chrispeels, 1976). Barley alsocontains both a proteinase that increases during germination and seedlinggrowth and an inhibitor of this proteinase (Mikola and Enari, 1970; Kirsi andMikola, 1971). The nature of this proteinase is unclear, but it may be one ofthe cysteine proteinases which are prominent in germinating barley (Koehlerand Ho, 1988;Marttila et aI., 1993). Avsenevaet al. (1988) noted the presenceof a com (Zea mays) seed proteinase inhibitor which inactivates a com serineproteinase.In each of these cases the level of inhibitor declines with germination and

seedling growth, while proteinase levels increase. The decline in inhibitorprecedes the increase in proteinase, and is generally complete before the pro-teinase activity has reached its maximum. The results suggest that the declinein inhibitor and the increase in proteinase are independent events, rather thanrepresenting the release of the proteinase from an inactiveproteinase /inhibitorcomplex due to destruction of the inhibitor component. Furthermore, in themung bean the increase in proteinase (vicilin peptidohydrolase) has beenshown to be due to de novo synthesi s (Chrispeels et aI., 1976). One possiblefunction of the inhibitors, suggested by Baumgartmer and Chrispeels (1976),is that they protect the cotyledonary cell in the event that one or more of theproteinase laden protein bodies rupture into the cytoplasm. Damage to thecell due to the released proteinase is thus minimized by its rapid reaction withthe inhibitor in the cytosol.There is one example where a proteinase / inhibitor complex may actually

serve as a transiently inactive reservoir of proteinase. This is the buckwheatmetalloproteinase / inhibitor system discussed above (see Section ILKabove).Confirmation of this case requires a more careful accounting of the relativeamounts of free and complexed proteinase and the exclusion of de novosynthesis of the proteinase.

B. Seed Proteinase Inhibitors as Protective Agents against ExogenousProteinases

It has been suggested that the proteinase inhibitors in seeds might serve toprotect the seed from microbial invasion or from the depredation of phy-tophagous insects (Ryan , 1973, 1979; Richardson, 1977; Birk, 1985). Forthis hypothesis to be seriously considered, it must be demonstrated that theproteinase(s) utilized by potential pathogens or insect pests are indeed sus-ceptible to inhibition by the inhibitor complement of the seed. It is perhapsobvious that one would not expect the proteinases of a pest (pathogen orherbivore) to be substantially inhibited by the proteinase inhibitors of thehost plant to which it is adapted. The pest, in adapting to the host, must have

356 Karl A. Wilson

overcome the defenses of the host. However, having stated this , it must alsobe pointed out that the host may still have minor inhibitors which, whileactive against the pest's enzymes, may simply be present in levels too low tomaterially disadvantage the pest . These inhibitors, while oflittle consequencein a natural ecological context, may provide important raw material for the'engineering' of resistant crop varieties (see Section IV below).The presence of inhibitors of microbial proteinases is widespread in seeds.

As noted above (Section II.F) many seeds contain inhibitors in the potato pro-teinase inhibitor I family that inhibit the bacterial serine proteinase subtilisin.Other inhibitors active against fungal and bacterial proteinases have been iden-tified in a number of plant seeds, especially through the efforts ofMosolov andcoworkers, e.g. in kidney bean (Phaseolus vulgaris) against the proteinasesof Fusarium solani, Aspergillus oryzae, and Colletotrichum lindemuthianum(Mosolov et aI., 1976, 1979, 1982, 1983); in com (Zea mays) against variousfungal proteinases (Halim et al., 1973; Mosolov et aI., 1984); in wheat, rye,and triticale (Mosolov et aI., 1976); in buckwheat (Fagopyrum esculentum)against Alternaria alternera proteinase (Dunaeskii et aI., 1994); and in bittermelon (Momordica charantia) against Streptomyces griseus endopeptidase(Ogata et aI., 1991). While the finding of these inhibitory activities is highlysuggestive of an antifungal/antimicrobial function for the inhibitors in theseed, no studies have appeared that directly link the presence, absence, orlevel of inhibitor with the seed's degree of resistance to invasion by thesemicrobes.The idea that the proteinase inhibitors of seeds serve a protective role

against insect pests is an attractive one, as it would explain the widespreadpresence of these proteins, and the apparent evolutionary conservation oftheir inhibitor functions . The utilization of proteinase inhibitors as defensiveagents in vegetative tissue has been well documented, especially through theefforts of CA. Ryan and his coworkers (Ryan and Green, 1974; Broadwayet aI., 1986). Some of the earliest support for this theory comes from thework of Birk and Applebaum (1960). Their interest stems in part from theobservation that the red and confused flour beetles iTribolium castaneum andT. confusum, respectively) are common pests in stored cereals, but not in rawsoybean meal. They demonstrated that the acetone insoluble trypsin inhibitorfraction (in large part the Bowman-Birk trypsin inhibitor) was at least partiallyresponsible for the growth impairment in the larvae of these coleoptera whenfed soybean meal (Birk, 1985). Birk et al. (1963b) subsequently demonstratedthat an inhibitor (apparently a minor component of the acetone insolublefraction) could be isolated from soybeans that inhibited Tribolium larval gutproteinases but was inactive towards bovine trypsin and chymotrypsin. Aninhibitor with a similar activity was also demonstrated in wheat (Applebaumand Konign , 1966). These results were in concordance with earlier work byLipke et al. (1954) that showed that trypsin inhibitors were not responsiblefor the growth inhibition of Tribolium on soybeans.


These results suggest that resistance to insect depredation is much morecomplex than just the presence or absence of trypsin or chymotrypsininhibitors. In part this is due to the variety of midgut proteinases foundin insec ts. Most early studies of insect gut proteases not unexpectedly uti-lized assay methods similar or identical to those used to study mammaliangut proteases. Thu s, for example, Applebaum et aI. (1964) found ' trypsin'and 'carboxypeptidase B ' in Tenebrio molitor. However, it has now beenshown that not all insects utili ze serine endopeptidase as their major diges-tive proteinases. Lepidoptera, Orthoptera, and Diptera appear to utilize serineproteinases in their digestive tracts (Broadway and Duffey, 1986; Murdock etaI., 1987; Larocque and Houseman, 1990; Chr isteller et aI., 1990, 1992).In contrast, the digestive apparatus of many Coleoptera, especially seed-

eating groups such as the bruchid beetles, use cysteine proteinase(s) (Gate-house et aI., 1985; Murdock et aI., 1987,1988; Wieman and Nielsen, 1988;Hines et aI., 1990; Silva and Xavier-Filho, 1991) and aspartic proteinases(Silva and Xavier-Filho, 1991). One may hypothesize that by adopting cys-teine proteinases as their major gut proteinases, these beetles have avoided theeffects of the plant seed's defensive serine proteinase inhibitors. This relianceon cys teine proteinases explains the poor correl ation between the levels oftrypsin inhibitors in various cowpea (Vigna unguiculata) cultivars and theirresistance to the bruchid beetle Callosobruchus maculatus (Xavier-Filho etaI., 1989), and also the failure of soybean trypsin inhibitors to retard thegrowth of Tribolium (see above). As the phytocystatins genera lly appear tobe prese nt in relatively low levels in seeds (at least compared to the serineproteinase inhibitors) they are readily overcome by the excess of cysteineproteinases in adapted insects.

C. Seed Proteinase Inhibitors as Storage Proteins

The proteinase inhibitors often constitute a significant portion of the solubleprotein of the seed (Ryan, 1973). In legumes, such as the soybean, the trypsinand chymotrypsin inhib itors may represent up to 6% of the protein (dependingupon spec ies , cultivar, and growth conditions), while in barley they representup to 10% of the protein. During germination these inhibitors disappear fromthe seed, generally with time courses similar to those observed for the bulkstorage protein (Pus ztai, 1972 ; Roy and Singh , 1988;Mikola and Enari , 1970;Salmia, 1980; Ikeda and Kusano, 1978; Xavier-Fi1ho and Negreiros, 1979;Yoshikawa et aI., 1979). Pusztai ( 1972) first proposed that in the commonbean (Phaseolus vulgaris) the trypsin inhibitors in fact act as storage proteins,espec ially as a reduced sulfur dep ot.Th is idea is especially attractive for the Bowman-Birk type inhibitors. As

noted above (Section II.C), inhibitors of this family contain approximately20 residu e% half-cystine. These inhibit ors thus represent a significant por-tion of the sulfur-containing amino acids of the seed, where the bulk of the

358 Karl A. Wilson

storage globulins (vicilins and legumins) are relatively poor in half-cystineand methionine. The same argument applies to the inhibitors of the Kunitz,squash trypsin, mustard trypsin, CM-protein/napin, and maize bifunction-al/thaumatin families . All of these contain relatively high contents of half-cystine and could thus serve as important seed sulfur depots.The degradation of the mung bean trypsin inhibitor (MBTI) and of the

soybean Bowman-Birk (BBSTI) and Kunitz (KSTI) trypsin inhibitors havebeen extensively characterized (Wilson 1988). In the mung bean, the inhibitorundergoes a series of cleavages that initially produce a series of identifiableintermediates (Lorensen et aI., 1981; Wilson and Chen , 1983). The cleavagesare initiated by a highly specific serine proteinase, proteinase F (Wilson andTan-Wilson, 1987), and then continued by two serine carboxypeptidases (Iand II) (Wilson et al., 1985) and at least one other endopeptidase. In thesoybean the initial degradation of both BBSTI and KSTI involve truncationsat the carboxyl-terminus (Hartl et aI., 1986; Madden et aI., 1985). Both ofthese reactions are catalyzed by the cysteine proteinase K I, which peaks inactivity four days after imbibition of the seed (Wilson et al., 1988; Papastoitsisand Wilson, 1991). At least two other proteinases (K2 and K3) have beenidentified that further degrade KSTI (Wilson et aI., 1988).It seems likely that in many instances the proteinase inhibitors may play

multiple roles in the seed simultaneously, or different functions at differenttimes during the life of the seed. Thus, during seed development, maturation,and quiescence some inhibitors (such as those of serine and cysteine pro-teinases) may protect the seed from insect depredation and microbial attack,either on the mother plant or in the soil. Later, during seed germination andearly seedling development, these same inhibitors are degraded to supplyamino acids and reduced sulfur (as cysteine) to the seedling until photosyn-thetic autotroph ism is established. In other cases the inhibitor found in themature seed may have no particular function at that stage. Instead, its pres-ence may be a vestige of its prior function in the developing seed, where itserved to prevent premature degradation of the storage proteins during syn-thesis, processing, and packaging into the protein bodies (Le. by regulatingendogenous processing proteinases).

IV. Future Directions

Future work on the seed protease inhibitors is likely to take several interrelatedtracks. The application of the techniques of molecular biology will yieldincreasingly more data on the nature and regulation of expression of theinhibitor genes. The protease inhibitors also provide an attractive means toincrease the resistance of crop plants to insect depredation. The production oftransgenic plants constitutively expressing sufficient levels of the appropriateinhibitor(s) would circumvent a number of problems in insect control. Such


control would be continuously present in the plant and relatively unaffectedby weather conditions. In contrast, the chemical insecticides presently usedmust be reapplied periodically, with the success of the application and itseffective lifetime subjec t to weather conditions. Furthermore, the use of theprotease inhibitors would avoid most of the undesirable toxic side effects ofchemica l insecticides, the proteins being relatively non-toxic to vertebrates,desirable poll inators, etc. In addition, they are readily biodegraded when theyare released into the environment upon the death of the plant.Several examples of the production of insect resistant transgenic plants are

already available. Tobacco plants transformed with a chimeric gene consist-ing of the strong con stitutive cauliflower mosaic virus 35S promoter and theKun itz soybean trypsin inhibitor exhibit resistance to the tobacco tudworm(He licoverpa assulta) (Koo et al., 1992). Tobacco plants transformed toexpress tomato proteinase inhibitor II have increased resistance to tobac-co homworm (Manduca sexta) larvae (Johnson et al., 1989), while plantsexpress ing cowpea trypsin inhibitor retard the growth of a number of lepi-dopteran pests including M . sexta , tobacco budworm (Heliothis virescens),H. zea, Spodoptera littoralis , and Autographa gamma (Hilder et al., 1987).The selection of the appropriate inhibitor for the transformation of a crop

plant is not necessarily trivial, and depends upon the digestive proteases of theinsects agai nst which resistance is sought. An inappropri ate inhibitor will notconfer the desired resistance. Transgenic tobacco plants expressing tomatoinhibitor II have increased resistance to M. sexta, while plants expressingtomato inhibitor I have little effec t on the larvae (Johnson et al. 1989). Thedifference is apparently due to the very different inhibitory specificities of thetwo tomato inhibitors: I is a strong chymotrypsin inhibitor and a weak trypsininhibitor, while II is a strong inhibitor of both trypsin and chymotrypsin.An example of a logical approach to the selection of the appropriate

inhibitors for tran sgenic manipulation has been taken by Christeller et al.( 1989) . Their long term goal is producing resistance to the grass grub, Coste-lytra zealandica, a coleopteran larva, in pasture grasses. They have first char-acterize d the dige stive enzymes of the insect (Christeller et al., 1989), andthen screened a panel of 26 inhibitors from ten inhibitor families againstthe major protease (a trypsin) (Christeller and Shaw, 1989). These inhibitorsexhibited a wide range of dissoci ation equilibrium constants (Kj), suggestingthat the choice of inhibitor is important.This observation lead s to another path of research - the characterization

of new protease inhibitors. The work on seed protease inhibitors to date hasbeen very much biased toward inhibitors of serine proteinases in legume andcereal crops, and occasionally in wild species in these families (e.g. Janzenet al., 1986). Whil e a few inhibitors from a few other plant families havebeen examined as noted above, the inhibitory complements of most plantfam ilies are unknown. Future research should address this weakness, both forthe basic knowledge of the biology of the inhibitors it will afford, and also

360 Karl A. Wilson

for the increased range of inhibitors that will be made available for molecularengineering of crop plants .

Acknow ledgment

The preparation of this chapter was supported in part by a grant from theNational Science Foundation, DCB90l7420.

Refere nces

Abe, K., Emori , Y., Kondo, H., Suzuki , K., and Arai , S. (1987 a) Molecular cloning of a cysteineproteinase inhibitor of rice (oryza statin). Homology with animal cystatins and tran sientexpression in the ripening process of rice seeds. J. BioI. Chern . 262 : 16793-16797.

Abe, K., Kondo, H., and Arai , S. (1987b) Purification and characterization of a rice cysteineprote inase inhibito r. Agric. BioI. Chern . 51: 2763-2768.

Abe, K., Kondo, H., Watanabe , H., Emori, Y., and Arai , S. (1991) Oryzastatins as the firstwell defined cystatins of plant origin and their target proteinases in rice seeds. Biomed.Biochim. Acta 50: 637-641.

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[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || The Protease Inhibitors of Seeds