Proteolytic degradation of cereal prolamins—the problem with proline

Plant Science 161 (2001) 825– 838 www.elsevier.com/locate/plantsci

Review

Proteolytic degradation of cereal prolamins —the problem with proline David J. Simpson * Carlsberg Laboratory, Department of Physiology, Gamle Carlsberg6ej 10, DK-2500 Valby, Denmark Received 28 March 2001; received in revised form 13 June 2001; accepted 14 June 2001

Abstract Cereal storage proteins serve as a store of amino acid nitrogen for the development of the embryo during germination. They are unusual in being susceptible to proteolytic degradation in their native conformation, in spite of their high percentage of the imino acid proline, which forms peptide bonds that are cleaved only by specialized peptidases. During germination, the endosperm is acidified to pH 5 by organic acids secreted from the aleurone, and is made reducing by the secretion of cysteine/glutathione and thioredoxin h/NADPH into the endosperm. These processes establish an optimal pH for proteolysis and help solubilize prolamins that are cross-linked by disulfide bonds. The initial cleavage is carried out by cysteine endoproteases secreted by the aleurone into the endosperm in response to gibberellins from the embryo. This group of cysteine endoproteases is unique to cereals, and may have evolved from a more widely spread group that are involved in protein degradation during programmed cell death. Studies with recombinant wild type and site-directed mutants of C hordein from barley reveal that primary cysteine endoprotease cleavage sites are located mainly in N- and C-terminal domains. This suggests a model in which removal of the C hordein terminal domains is necessary for unfolding of the b-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack at secondary sites. Proteolysis of the resulting peptides is carried out by serine carboxypeptidases, most of which are synthesized de novo in the scutellum and aleurone during germination, and are secreted into the endosperm. The presence of proline next to the first and/or last residue protects peptides against attack by the normal exopeptidases and requires the action of proline-specific peptidases. The peptides and amino acids produced by proteolysis in the endosperm are transported into the embryo where di- and tri-peptides are further degraded to amino acids. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Carboxypeptidase; Cysteine endoprotease; Fluorogenic substrates

1. Introduction The major storage proteins of cereals stored in the endosperm tissue of the kernel are the prolamins, so named because of their high content of proline and glutamine (see review by Shewry [1]). Prolamins are also called hordeins (barley), zeins (maize), gliadins (wheat), avenins (oat) and secalins (rye). As a conse-

Abbre6iations: Abz, 2-aminobenzoyl; AMC, 7-amino-4-methylcoumarin; DFP, diisopropyl fluorophosphate; Dnp, dinitrophenyl; Tyr(NO2), 3-nitrotyrosine; Z, benzyloxycarbonyl. * Tel.: + 45-3327-5231; fax: + 45-3327-4765. E-mail address: [email protected] (D.J. Simpson).

quence of their unusual amino acid composition, they are insoluble in water. This property, combined with the abundance of proline could cause problems for cleavage by endoproteases, since proline cannot form hydrogen bonds and there is limited rotation about the prolyl peptide bond. Nevertheless, proteolysis of prolamins during germination is the result initially of endoprotease activity, and the resulting oligopeptides are substrates for exopeptidases, which remove amino acids sequentially from their amino or carboxy termini. The proteolytic enzymes responsible for this process are either synthesized and stored in the endosperm during grain development, or are synthesized and secreted during germination from the aleurone or scutellum, which completely surround the endosperm. Because the

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cereal starchy endosperm undergoes programmed cell death during grain maturation, internal cellular compartmentalization is destroyed. This implies the absence of membrane barriers between prolamins in the endosperm and the resident proteases, or those secreted into the endosperm in the germinating grain. The mixture of amino acids, di-, tri- and oligo-peptides resulting from prolamin proteolysis is determined mainly by the substrate specificity of the exopeptidases present in the endosperm. Di- and oligo-peptides of up to five amino acid residues in the endosperm can be assimilated by the scutellum epithelium cells of the developing embryo by a single uptake system that is specific for L-amino acid residues and requires a proton gradient [2]. There is evidence for several amino acid uptake systems, including one specific for proline [3 – 5]. Oligopeptides taken up by the scutellum are further degraded to single amino acids, and in view of the amino composition of prolamins, the exopeptidases involved are probably proline-specific. The acidification of the starchy endosperm during germination by organic acids secreted by the aleurone requires that proteolysis in the endosperm is carried out by proteases which are most active between pH 4– 5.5, whereas the environment of cytoplasmic scutellar peptidases is likely to be neutral or alkaline (pH 7– 9).

the gel in specific inhibitors, and comparing the activity zones with and without inhibitor. This technique has been used by Jones and co-workers [6] to demonstrate the presence of 42 endoproteases in green barley malt, including 27 cysteine endoproteases, seven serine endoproteases, four metalloproteases and four aspartate endoproteases. Although some of these are isoforms, this large number of proteases does not include exoproteases, which are not detected by this technique. Endoproteases that are inactive in the above assays can be detected with colorimetric or fluorescently labeled protein substrates, such as azocasein or casein fluorescein isothiocyanate (Sigma St, Louis, MO). The presence of low levels of active endoproteases can also be detected more sensitively with BODIPY FL or BIDIPY TR-X fluorescently labeled casein (Molecular Probes, Eugene, OR), or the Pep-Tag protease assay system from Promega (Madison, WI), where cleavage is detected by a change in pI. Having established the presence of a protease in a crude extract, a more convenient assay is needed to follow its purification through a series of chromatographic steps. The most easy-to-use protease substrates are those which are specific for the peptidase of interest. One class of such substrates has the formula: (A)n –Y

2. How to detect and assay for protease activity The detection of new or uncharacterized endoproteases is best achieved using non-specific substrates such as gelatin, hemoglobin and edestin, to increase the likelihood of cleavage and detection. If the protein substrate is immobilized in SDS-polyacrylamide gels (SDS-PAGE), these can be used as activity gels to detect endoproteases and give an estimate of their molecular weight, provided that the protease is not sensitive to the presence of SDS, or can be renatured after denaturation by SDS. When looking for endoproteases involved in prolamin degradation, the relevant prolamin can be used, although this has rarely been done. After electrophoresis, the gel is incubated in the appropriate buffer(s), usually including Triton X100 to promote the removal of SDS, and subsequently stained with Coomassie blue [6]. Proteolysis of the immobilized substrate creates a clear zone in a dark blue background. If the protease is active during electrophoresis (e.g., subtilisin or protease K), the electrophoretic lane will be clear down to the final migration distance of the protease. The use of two-dimensional gel electrophoresis, where the first dimension involves protein separation by isoelectric focusing, not only gives better resolution, but also the pI of the protease(s). Information about the catalytic mechanism of the endoprotease(s) can be obtained by incubating

where A is any amino acid and Y is a chromatophore or fluorophore attached to the C-terminus which can be detected spectrometrically after proteolysis. These substrates require the specific cleavage of the peptide– chromatophore bond, but the chromatophore is not always accepted in the S1% subsite of the protease substrate binding site (Fig. 1, the enzyme binding sites are denoted Si, …S2, S1 and S1% , S2% ,…Sj% away from the scissile bond between S1 and S1% . The substrate positions are denoted Pi,…P2, P1, P1% , P2% , …Pj% corresponding to the enzyme binding sites [7]). The 7-amino-4-methylcoumarin peptide substrates also require a specific cleavage of the fluorochrome-peptide bond (Fig. 2a). If cleavage occurs at another site in the peptide moiety, it

Fig. 1. Diagram of a protease substrate binding site. Residues of the substrate are indicated P4, P3 etc, with cleavage occurring between P1 and P1% , indicated by the arrow. Each residue fits into a corresponding binding subsite (S4, S3 etc.) in the protease.

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aminobenzoyl and 3-nitrotyrosine [8], and Edans (5(2-aminoethyl)aminonaphthalene-1-sulfonic acid) and Dabsyl (4-(4-dimethylaminophenylazo)-benzoic acid) [9]. These types of substrates are particularly suitable for the determination of endoprotease substrate specificity, but few are commercially available.

3. Initial prolamin degradation by cysteine endoproteases

Fig. 2. Chemical structures of protease substrates. (A) Only cleavage of the peptidyl bond between the aminomethylcoumarin group and the attached amino acid or peptide results in a change in the excitation and emission spectrum of the product. These are good aminopeptidase substrates, but may not be good endoprotease substrates if the aminomethylcoumarin group is poorly accommodated at S1% . (B) Internally quenched, fluorogenic peptide substrates consist of a peptide sequence flanked by a fluorochrome (e.g. 2-aminobenzoyl) and a quenching group (e.g. 3-nitrotyrosine). Cleavage at any peptide bond relieves the distant-dependent quenching, and the presence of residues at P1% , P2% , P3% etc. can greatly increase substrate specificity.

will not be detected fluorometrically. Moreover, if endoprotease specificity is significantly affected by the nature of the residue at P%1, these compounds may be very poor substrates. They are, however, commercially available, sensitive, relatively inexpensive, and are particularly useful for assaying aminopeptidases. A more recent development has been the introduction of intramolecularly quenched fluorogenic peptide substrates, where the detection of cleavage is not dependent on the position of the scissile bond. These substrates have the general structure: X –(A)n –Y where A is any amino acid, X is the fluorescing group, and Y is the quenching chromatophore (Fig. 2b). Fluorescence quenching involves resonance energy transfer over a distance that is dependent on the spectral overlap between the donor (X) and acceptor (Y). Cleavage of any peptide bond between donor and acceptor results in an increase in fluorescence. Suitable donor and acceptor pairs include o-

Identification of an endoprotease as being essential for prolamin degradation in cereal endosperm should satisfy the following prerequisites: (1) it must be present with its natural substrate in the same cell; (2) the time course of protease activity must coincide with that of prolamin degradation; and (3) it should degrade its natural substrate in vitro (after [10]). Although some proteases are deposited in the endosperm during development, the major ones are secreted from the aleurone or scutellar epithelium during germination. Those endoproteases that enter the secretory pathway are synthesized as pre-proenzymes, with a signal peptide to effect their transport across the endoplasmic reticulum, and an N-terminal propeptide, which must be removed before the protease is activated. Endoproteases are secreted if they lack vacuolar targeting sequences. In addition, C-terminal extensions, or telo sequences, may be required for correct folding or targeting [11]. Although there are many endoproteases in germinating grains, they may serve functions unrelated to storage protein degradation. Bioinformatics of the recently sequenced Arabidopsis genome, provides a valuable basis for examining the diversity of endoproteases in plants. The Arabidopsis thaliana genome encodes 32 papain-type (C1 family) cysteine endoproteases, which can be placed into eight main groups based on sequence similarity to other cysteine endoproteases (Fig. 3). Cereal cysteine endoproteases are members of six of these groups. Those known to be involved in prolamin degradation form a small, separate group, which does not include any A. thaliana members, but are most similar to endoproteases with a C-terminal KDEL motif (Fig. 3). This latter group is unique to plants and contains cysteine endoproteases that have recently been located to precursor protein vesicles of Ricinus communis that activate and burst during programmed cell death [12]. Presumably, the prolamin-degrading cysteine endoproteases have a non-functional KDEL-like motif at the C-terminus since they are not retained in the ER lumen, but are secreted into the endosperm. None of

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the other cysteine endoproteases identified in cereals are secreted proteins, so they are unlikely to be involved in prolamin degradation. Cysteine endoproteases are the most important group of proteases in germinating cereal grains, accounting for up to 90% of the total activity in degrading prolamins, based on class-specific inhibitor studies with barley [13], maize [14] and wheat [15]. Much of the cysteine endoprotease activity in barley is due to two proteases designated EP-A [16] and EP-B [17]. They are secreted into the endosperm during germination from the scutellar epithelium and aleurone in response to gibberellic acid [18,19]. EP-A is found as three isoforms, due to heterologous processing at the N-terminus, with a molecular mass of 37 kDa. EP-B, which is more abundant, has a molecular mass of 30 kDa, and occurs as two isoforms with pIs of 4.6– 4.7 [17,18].

Fig. 3. Phylogenetic relationship between C1 family cysteine endoproteases from Arabidopsis thaliana and cereals. The phylogenetic tree was drawn using the neighbor-joining method using the program Clustal X. At, Arabidopsis thaliana; Os, Oryza sati6a; Hv, Hordeum 6ulgare; Zm, Zea mays; Sc, Secale cereale; Sb, Sorghum bicolor. Accession numbers are shown in the figure except for At Sag12 (AT5g45890), At rd19 (AT4g39090), At Sag2 (AT5g60360), Os oryzain g (D90408), Hv aleurain (X05167), Zm Ccp2 (D45403), Zm See1 (X99936), At CEP1 (AT5g50260), At CEP2 (AT3g48350), At CEP3 (AT3g48340), Os RepA (X80876), Hv EPA (Z97023), Hv EPB (U19359), Os Rep1 (d76415), Os oryzain b (D90407), Os oryzain a (D90406), Zm Mir3 (AF019147), At rd21 (D13043), Zm Mir2 (AF019146) and Zm Mir1 (AF019145). Zm CP10A is from [14].

Fig. 4. Diagram of suggested C hordein secondary structure, showing central b-reverse turn helix of the proline and glutamine-rich octameric repeats flanked by globular domains. The primary cleavage sites of the cysteine endoprotease EP-B are indicated by arrows. It is proposed that the release of these domains enables the helix to unwind and expose secondary cleavage sites to endoprotease attack.

Proteases closely related to EP-A are found in rice and wheat, and EP-B orthologs are found in rice and maize (Fig. 3), and may have a similar function. In the proposed model for storage protein degradation in dicots such as Vigna mungo, Vicia sati6a and Phaseolus 6ulgaris, which occurs in living tissue, the first cysteine protease to act is synthesized de novo and produces a limited hydrolysis [10]. This generates new sites for a second type of cysteine endoprotease, which is inactive against the native substrate. In order to study barley prolamin degradation in detail, Davy et al. [20] expressed a single hordein polypeptide in E. coli as the substrate for proteolytic degradation. Although the major barley storage proteins are the sulfur-rich B hordeins, which have proline/glutamine repeat domains at their N-terminus, C hordein was chosen because it lacks cysteine and does not form intermolecular disulfide bonds, and the recombinant protein can be renatured to the native conformation in vitro [21]. It is thought that the proline-rich octameric repeats (PQQPFPQQ) in C hordein form a central b-reverse turn helix, flanked and perhaps stabilized by N- and C-terminal globular domains (Fig. 4). The three dimensional conformation of this molecule, together with its high proline content, makes it a challenging endoprotease substrate. The hydrolytic specificity of a 30 kDa [22] and a 31 kDa [23] barley cysteine endoprotease (which may be isoforms of EP-B) was earlier determined using nonhordein polypeptides such as hordothionin, purothionin and levitide. Davy et al. [20] purified EP-B and incubated it with recombinant C hordein and found several discrete bands, which were subsequently degraded to short oligopeptides after longer incubation. The individual polypeptide bands were subjected to N-terminal amino acid sequencing to determine the cleavage points from the known sequence of C hordein. These primary cleavage sites were FR¡QQ, FQ¡QP, VQ¡QP, LQ¡QP and LQ¡SP and are mainly found near the N and C-termini (Fig. 4). Numerous secondary cleavage sites in C hordein were deduced from the short oligopeptides that were separated by HPLC and identified by mass spectroscopy and amino acid sequencing. The most common site was PQ¡QP, which is a common motif

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found in the octameric repeat region of C hordein. No cleavage was detected in C hordein with proline at P1 or P1% . The substrate specificity of EP-A and EP-B has been determined systematically by using two series of fluorogenic peptide substrates in which the residue at P2 or P1 is varied [20]. The best substrate based on the C hordein primary cleavage sites (FRQQ) was used as the starting point, and 18 other residues were substituted for Phe at P2 (Table 1). These studies showed that EP-B (and EP-A) preferred neutral amino acids with large aliphatic and non-polar (Leu, Val, Ile, Met) or aromatic (Phe, Tyr, Trp) side chains at P2. A second series was made with Leu at P2, and Pro at P2% to direct cleavage between Xaa and Gln, and all 20 possible residues at P1 (Table 1). These results showed less specificity at P1, although Asn, Asp, Val and Ile were particularly unfavorable and peptides with Pro at P1 or P1% were extremely poor substrates. Substrates based on the most frequently cleaved secondary site, PQQP, where also cleaved extremely slowly, even when additional residues, based on the C hordein sequence, were incorporated at P3 and P3% . Site-directed mutagenesis of C hordein where Leu at P2 was replaced with Ser or Pro was shown to destroy primary EP-B cleavage sites [20]. When all the primary cleavage sites in C hordein were removed by site-directed mutagenesis, the resulting protein was degraded more than 100 times slower than wild type C hordein Table 1 Kinetic constants kcat/Km (mM−1 s−1) for the hydrolysis of substrates with substitutions at the P2 and P1 positions by the barley cysteine endoprotease EP-B P2P1¡P%1P%2

P2P1¡P%1P%2

Xaa–Arg¡Gln–Gln

Leu–Xaa¡Gln–Pro

Leu Phe Tyr Trp Val Met Ile Cys Arg His Thr Asp Glu Gln Ala Lys Asn Gly Ser

16 300 9 510 8580 9 100 5650 9 190 1200 9120 983 933 875 930 532 94 89 95 57.9 9 1.2 52.3 9 1.6 16.8 90.8 16.6 9 0.8 14.6 91.2 11.4 9 1.5 9.7 90.3 9.0 90.7 9.0 90. 7.2 9 0.4 3.3 90.1

Arg Met Leu Phe Tyr Gln Thr Lys Glu Gly Ala Ser Cys His Trp Asn Asp Val Ile Pro

9230 9 440 7500 9330 7270 9 410 7270 9 320 63609 260 5960 9 100 59309 110 5920 9 200 5340 9120 4260 9130 3950 9130 3350 9140 24909130 1320 940 1220 9 50 838 910 690923 588 931 372 921 N.D.

¡ indicates cleavage site N.D., not detected (B3), from [24].

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[24]. In view of the proposed structure of C hordein, and the location of the primary cleavage sites (Fig. 4), it was suggested that removal of the C hordein terminal domains is necessary for unfolding of the b-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack at secondary sites. The addition of b-mercaptoethanol in vitro increases the rate of hordein degradation by cysteine endoproteases, but is not essential. This effect is partly due to activation of the protease, and partly to the reduction of hordein disulfide bonds. The cleavage of intermolecular disulfide bonds in particular, may increase the solubility and accessibility of hordeins to proteolytic attack during germination [24]. This probably involves reduction by thioredoxin h and NADPH during germination [25] or by glutathione and cysteine [26], which are secreted by the embryo. In contrast to the model for storage protein degradation in dicots, it would appear that EP-B carries out the function of both of the dicot cysteine endoproteases. Isolated cereal cysteine endoproteases can extensively degrade prolamins in vitro, either in the form of native protein bodies from barley [24] or maize [27] or solventextracted wheat gliadin [28] and rice glutelin [29]. Legumains (asparaginyl endopeptidases) have been isolated from germinating rice (REP-2) and wheat [28], and have been suggested to be involved in the initial degradation of prolamins, but this remains to be confirmed experimentally.

4. Prolamin degradation by other endoproteases Serine endoproteases have not been extensively studied in plants, although they are among the best characterized of the mammalian endoproteases. A serine protease of the subtilisin family (subtilase) has recently been isolated from barley malt [30], and identified by N-terminal amino acid sequencing and labeling with [14C]-DFP. Hordolisin has an apparent molecular mass of 74 kDa, a pI of 6.9 and a pH optimum of 6.0, but does not appear to be involved in hordein degradation. This is in contrast to WEP-2, a serine endoprotease from germinating wheat grains, which degrades gliadin efficiently [28]. A 14 kDa serine protease capable of cleaving wheat gliadins and glutenins is activated by thioredoxin and calcium. This protease, named thiocalsin, exists as three isoforms with similar pH optima (pH 5.0) and is reported to be active during germination [31]. An aspartic endoprotease has been reported to be involved in the degradation of wheat gliadins (v and g) during germination of wheat grains [32] and to accelerate the proteolysis of gliadins by a crude cysteine endoprotease preparation [33]. Aspartic endoproteases

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have an acidic pH optimum and are inhibited by pepstatin A. They have a highly conserved three-dimensional structure with an extended substrate-binding cleft (from S4 to S3% ) containing two conserved Asp residues essential for catalytic activity. An aspartic endoprotease has been purified from barley [34] and rice [35] and the sequence determined. The barley enzyme (HvAP) is produced in the embryo and aleurone layer of the developing grain, but disappears from the aleurone during grain maturation [36]. It is synthesized during germination and accumulates in the protein bodies and large vacuoles of the aleurone and scutellum, and is not secreted into the starchy endosperm [37]. HvAP shows no activity towards denatured a-hordothionin or barley globulins [38] and the corresponding rice protease is inactive at pH 5.5, which is the pH of the protein storage vacuole during germination [35]. There is, therefore, no evidence that aspartic endoprotease plays a significant role in the proteolysis of endosperm storage proteins during germination. In vitro evidence indicates that it might be involved in C-terminal trimming of pro-peptides during transport of precursors to the vacuole and may participate in the hydrolysis of storage proteins in the embryo and aleurone [35]. Aspartic endoprotease may also be involved in the processing and activation of protease zymogens, which are secreted from the aleurone [39]. Metalloendoprotease activity has been reported in barley malt and resolved into up to five different fractions with apparent molecular masses of 122, 164, 179, 224 and \ 220 kDa [40]. They were detected by their ability to cleave gelatin and their activity was inhibited by EDTA and restored by the addition of Zn++ or Mg++. They are active at relatively high pH and account for between 7 and 10% of total endoprotease activity in barley malt [13,41]. None of the cereal metalloendoproteases has been purified or characterized. A low molecular mass (34 kDa) metalloendoprotease involved in the proteolysis of 13S globulin has been purified from buckwheat (Fagopyrum esculentum) [42].

5. Cleavage of prolamin fragments by serine carboxypeptidases Serine carboxypeptidases are the major exopeptidases found in germinating cereal grains, responsible for the production of about 75% of the free amino acids during brewery mashing [43]. Chromatographic separation of protein extracts from germinating barley [44] and wheat grains [45] has resolved five fractions with serine carboxypeptidase activity, suggesting the existence of at least five different serine carboxypeptidases, designated CP-MI, CP-MII, CP-MIII, CP-MIV and CP-MV. The

Table 2 Hydrolysis of Z–Ala–X–OH and Bz–Y–OMe substrates by serine carboxypeptidases Substrate

kcat/Km (min−1 mM−1) CP-MI

CP-MII

CP-MIII

P %1 Z–Ala–Gly Z–Ala–Ala Z–Ala–Val Z–Ala–Ile Z–Ala–Met Z–Ala–Phe Z–Ala–Pro Z–Ala–Asp Z–Ala–Asn Z–Ala–Lys Z–Ala–Arg Z–Ala–His Z–Ala–Ser

480 22 000 35 000 22 000 18 000 5700 2600 370 270 B5 B5 120 1080

11 560 1800 3300 3200 2100 6 71 62 25 000 18 000 1100 150

11 2100 17 000 27 000 43 000 94 000 120 B1 B1 7 9 1 5

P1 Bz–Gly–Ome Bz–Ala–Ome Bz–Val–Ome Bz–Ile–Ome Bz–Leu–OMe Bz–Met–OMe Bz–Phe–Ome Bz–Asp–Ome Bz–Lys–Ome Bz–Arg–Ome Bz–His–Ome Bz–Thr–OMe Bz–Pro–OMe

B5 370 22 17 420 1200 14 600 B5 18 300 25 300 5600 69 B5

B5 19 7 8 310 160 5700 B5 2200 950 76 B5 B5

B1 200 35 34 820 750 1000 2 3 18 23 5 B1

From [47].

first three have been isolated from barley malt and characterized as to substrate specificity [46–49]. CPMII is the only serine carboxypeptidase that is expressed and accumulated in the developing grain, and is stored in an active form in the mature grain [50]. CP-MI, CP-MII and CP-MIII are synthesized de novo in the scutellum and aleurone during germination, and are secreted into the endosperm. cDNAs encoding three additional CP-MII isoforms have been sequenced from barley, and since they differ from CP-MII at residues in the substrate-binding subsites, they may have different substrate specificities. Substrates with aspartic acid or glutamic acid at their C-terminus are very poorly hydrolyzed by the purified barley serine carboxypeptidases (Table 2), so the presence of Arg-485 at the S1 subsite of one of these CP-MII’s may indicate an affinity for such substrates [50]. CP-MI and, to a lesser extent CP-MIII, will cleave proline at the C-terminus (Table 2), but not when proline is in the penultimate position. However, extensive studies of yeast carboxypeptidase, which resembles barley CP-MIII, have shown that substrate length and

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the P1 –P5 substrate sequence can affect kcat/Km values by up to a factor of 6 × 107 [51]. Thus kcat/Km for the substrate AbzAAGGP¡Y(NO2) is 2.3, whereas for AbzFFLFP¡Y(NO2) it is 160000 (min − 1 mM − 1). Nevertheless, most peptides with proline in the penultimate position are extremely poor substrates for CP-MI, II and III. 6. Other exoproteases An extensively characterized thiol exoprotease of germinating cereal grains is the aminopeptidase aleurain, from barley [52], and homologous proteases have been sequenced from rice [53] and maize [54]. Barley aleurain is constitutively expressed in most plant tissues, but its synthesis in the aleurone layers is regulated by the plant hormones gibberellin and abscisic acid. It is synthesized as an inactive precursor during germination in response to gibberellic acid, and is stored in small discrete vacuoles in the aleurone [55], indicating that it is not involved in the proteolysis of prolamins. Another thiol aminopeptidase of approximately 65 kDa has been partially purified from barley malt, and is inhibited by p-chloromercuribenzoate but not affected by DFP or EDTA [43]. It showed a preference for large hydrophobic amino acid residues in dipeptide and Laminoacyl-b-naphthylamide substrates. Charged residues (Glu, Arg, Lys) and glycine were very poor substrates, while some activity was found for proline. Its pH optimum with dipeptide substrates was 5.8–6.5, indicating its possible involvement in peptide hydrolysis in the endosperm during germination. Similar thiol aminopeptidases have been reported in maize and wheat.

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Not many plant metallopeptidases have been reported, and even fewer have been purified and characterized. A leucine aminopeptidase has been partially purified from germinating barley grains [56], and is similar to mammalian and other plant leucine aminopeptidases. The barley grain enzyme is probably a homohexamer with a molecular weight of 260 kDa and a pH optimum between 8.5 and 10.5. It is activated by Mg++ and Mn++, and is inactive in the absence of reducing sulfhydryl compounds, which is in contrast to the mammalian leucine aminopeptidases [56]. A leucine aminopeptidase inhibited by bestatin, but otherwise not characterized as to active site residues, has been purified from kidney bean cotyledons. It has a pH optimum of 9, is stabilized by dithiothreitol and its activity increased 3-fold by Mg++. It is the most abundant peptidase in kidney bean cotyledons and is thought to participate in the hydrolysis of reserve proteins [57]. The substrate specificities of the barley and bean leucine aminopeptidases are similar, with higher activity towards longer substrates. Leucine-r-nitroanilide is a poor substrate, as are those with proline in the second position (Table 3). A second barley metalloprotease has been purified and partially characterized from malt. The enzyme is inhibited by metal chelators and r-chloromercuribenzoate, indicating the involvement of both a metal ion and a sulfhydryl group at the active site. The substrate specificity showed that the enzyme is a specific dipeptidase, with no activity towards tripeptides or dipeptides lacking a free carboxy group [58]. The native enzyme has a mw of between 130 and 175 kDa, with a subunit mw of 50 kDa. The relative hydrolysis rates and Km values for some dipeptides are shown in Table 4.

Table 3 Substrate specificity of plant leucine aminopeptidases Substrate

Leu–Gly–Gly Leu–Gly Leu–Tyr Leu–NH2 Leu–b-NAc Met–Leu–Gly Ala–Pro–Ala Ala–Ala–Pro Ala–Leu Ala–Gly Phe–Phe Tyr–Glu Gln–Gln His–Gly a

From [56]. From [57]. c Leucyl–b-naphthylamide. b

Barleya

Kidney beanb

Relative activity (%)

Km (mM)

Relative activity (%)

Km (mM)

100 52 62 16 0.06 87 – – – 0.07 – – – –

– 9.6 0.16 – 0.16 0.16 – – – – – – – –

100 51 23 – – 32 B0.5 82 20 2 12 24 18 27

3.7 1.6 0.4 – – 1.0 – 3.3 – – 0.1 1.4 – 10

D.J. Simpson / Plant Science 161 (2001) 825–838

832 Table 4 Substrate specificity of barley dipeptidase Substrate

Relative rate (%)

Km (mM)

Ala–Gly Ala–Ala Ala–Val Ala–Leu Gly–Gly Val–Gly Leu–Gly Ser–Gly Tyr–Gly Pro–Gly His–Gly Lys–Gly Gly–Ser Gly–Leu Gly–Tyr Leu–Tyr Asp–Ala

100 63 16 102 0.7 32 6.2 9.5 8.5 0.9 0.2 B0.05 1.5 3.5 15.9 8.3 0.05

15.8 2.4 5.4 6.4 – 1.4 0.3 – – – – – – – – – –

From [58].

and/or which end with PQ, e.g. QPQ, QPFPQ and QPLPRPQ (Fig. 5). These are not substrates for the usual amino- and carboxypeptidases. Proline-specific peptidases have been mostly characterized from bacteria and mammals, but their presence in plants has been shown by activity measurements (proline iminopeptidase, prolyl carboxypeptidase and dipeptidyl peptidase IV) and from EST and genomic sequencing projects (Table 5). The properties and occurrence of known plant proline-specific proteases are summarized below and in Table 6. They cleave peptide substrates with different specificity, as shown in Fig. 6.

7.1. Aminopeptidase P Aminopeptidase P (also called proline aminopeptidase) is a metalloprotease, based on its inhibition by metal chelators, but is also inhibited by thiol functional reagents, indicating the presence of functionally important cysteine residues remote from the active site [59].

Dipeptides with basic or acidic residues in P1 (Lys, His, Asp) were very poor substrates, whereas those with neutral amino acids at P1 were hydrolyzed, including proline.

7. Cleavage of prolamin peptides by proline-specific peptidases The secondary structure of cereal prolamins is determined by their high content of the imino acid proline, requiring specialized proteases for their degradation during germination. Since the side chain of proline is bonded to both the amino group and the a-carbon, the molecule is circular. It has no functional groups, so it cannot form hydrogen bonds, and the limited rotation about the peptide bond introduces a reverse turn or hairpin bend into the peptide chain. Proline residues within polypeptide precursors often reduce their susceptibility to further proteolysis, particularly for the X– Pro peptide bond at the N-terminus, but also for Pro –X at the C-terminus. This implies that specific proteases have evolved to cleave the peptide bond on either side of proline residues, many of which have a high substrate specificity for proline at P1. The prolinespecific peptidases can be grouped into those which cleave X–Pro bonds (the metalloproteases aminopeptidase P and prolidase) and those which cleave Pro–X bonds (the serine peptidases prolyl oligopeptidase, prolyl iminopeptidase, dipeptidyl peptidase IV, prolyl carboxypeptidase and prolinase), where the term ‘prolyl’ refers to the cleavage of the prolyl bond. Cleavage of C hordein by the cysteine endoprotease EP-B occurs at the sequence PQ¡QP many times, resulting in peptides which begin with the sequence QP,

Fig. 5. C hordein amino acid sequence. The sequence appears twice and is read sequentially in each column, starting from the top. The first column shows minor (¡) and major (n) cysteine endoprotease cleavage sites and is aligned to show the octameric repeats. In the second column, each line ends at a proven cysteine endoprotease cleavage site and shows the frequency of proline (P) next to the first and/or last residue. Peptides with proline at these positions can only be cleaved by proline-specific exopeptidases.

D.J. Simpson / Plant Science 161 (2001) 825–838

833

Table 5 Proline-specific peptidases in Arabidopsis thaliana Peptidase

EC number

Type

MIPS protein entry code

Proline iminopeptidase Aminopeptidase P Prolyl carboxypeptidase Prolyl oligopeptidase DPP IV DPP II Prolidase Prolinase

EC EC EC EC EC EC EC EC

Serine Metallo Serine Serine Serine Serine Metallo Serine

At2g14260, At3g05350, At2g18080, At1g20380, At5g24260 None At1g09300, None

3.4.11.5 3.4.11.9 3.4.16.2 3.4.21.26 3.4.14.5 3.4.14.2 3.4.13.9 3.4.13.8

This peptidase cleaves before proline residues next to the amino terminus (Xaa – Pro – ), and is able to cleave Pro – Pro peptide bonds. The intramolecularly quenched substrate Lys– (Dnp) – Pro – Pro – Edans-Abz provides a sensitive and specific assay for aminopeptidase P, since it is not cleaved by prolyl carboxypeptidase or dipeptidyl peptidase. Gly– Pro –AMC is also an excellent substrate for this enzyme from mammals. Rat aminopeptidase P is reported to have optimal activity at pH 7.0–8.5, and to be inhibited by thiol functional reagents [59]. Mikola and Mikola [60] report cleavage of the dipeptide Ala– Pro by a 5-day scutellum extract at pH 5.1, which may indicate the activity of an aminopeptidase P (or a prolidase). Two genes for proline aminopeptidase have been identified in Arabidopsis thaliana (Table 3), and ESTs have been found in rice, wheat, barley and maize.

7.2. Prolidase Prolidase (or prolyl dipeptidase) removes N-terminal amino acids from dipeptides with proline at P1% (Fig. 6). In mammals, it is involved in the degradation of collagen and the subsequent recycling of proline [59]. Prolidase appears to be a metalloprotease and is a strict dipeptidase that cleaves dipeptides containing an N-terminal proline residue. Prolidase has been purified from germinating soybeans [61] and two genes have been identified in Arabidopsis (Table 3). ESTs have been found in barley, wheat, rice, maize and sorghum.

At3g61540 At4g36760 At2g24280, At4g36190 (2), At5g22860, At5g65760 At1g76140

At4g29490

appearance of most of the free proline in the autolyzing part of the starchy endosperm, based on the complete inhibition of this process by DFP [60]. Barley prolyl carboxypeptidases are unable to release proline from dipeptides, although Z-dipeptides, such as Z–Pro –Ala, Z–Pro –Met and Z–Pro – Trp, are substrates and Z– Gly –Pro – Ala is an even better substrate for one of the barley prolyl carboxypeptidases [44]. Prolyl carboxypeptidases were also purified from wheat, where they were found in resting and in germinating grains and in flag leaves [45]. Six prolyl carboxypeptidase genes have been identified in Arabidopsis thaliana (Table 3) and ESTs are known from barley, rice, wheat, sorghum and maize. Carboxypeptidases can be assayed by measuring the release of the C-terminal amino acid from free peptide substrates using an automated ninhydrin assay, or the formation of hydrolysis products by HPLC, both of which are time-consuming. Hydrolysis of 3-(2-furylacryoyl)-dipeptides (e.g. [62]) can be monitored by the increase in absorption (329–352 nm). Olesen et al. [51] have shown that carboxypeptidase Y can cleave a Cterminal nitrotyrosine residue, so that internally quenched fluorogenic peptide substrates with the general formula Abz– P5 –P4 –P3 –P2 –P1 –Tyr(NO2)–OH can be used to determine substrate preferences. Such substrates could provide a convenient means to monitor the purification of prolyl carboxypeptidases and to determine their substrate specificity.

7.4. Prolinase 7.3. Prolyl carboxypeptidase Prolyl carboxypeptidases cleave the C-terminal residue from proteins that contain proline in the penultimate position (Fig. 6). They are inhibited by metal chelators but a serine residue necessary for catalytic activity has been identified. Two prolyl carboxy-peptidases have been isolated from germinating barley grains [44,60]. These enzymes were inhibited by diisopropylfluorophosphate (DFP) and stabilized by 0.5 mM dithiothreitol, and had apparent molecular masses of 170 and 95 kDa. They appear to be responsible for the rapid

Prolinase, or proline iminodipeptidase, removes proline from dipeptides with proline at P1. It is a strict dipeptidase, but no ESTs coding for plant prolinases are published, nor has a prolinase gene been identified in the A. thaliana genome.

7.5. Proline iminopeptidase Prokaryotic proline iminopeptidase is a strict exopeptidase that can release the N-terminal proline from peptides of any length, and can cleave Pro–Pro bonds.

D.J. Simpson / Plant Science 161 (2001) 825–838

834 Table 6 Properties of plant proline-specific peptidases Peptidase

pH optimum

Cleavage of Pro–Pro

Cleaves

Substrate

Proline iminopeptidase Aminopeptidase Pa Prolyl carboxypeptidase Prolyl oligopeptidase DPP IV DPP IIa Prolidase Prolinasea

7.5 7–8.8 5.0 7.3 7.2 5.5 7.0 8.5

Yes Yes No No No Yes ? ?

NH2–Pro -X– NH2–X -Pro–Y Acyl–X–Pro -Y Acyl–X–Pro -Y NH2–X–Pro -Y NH2–X–Pro -Y5 NH2–X -Pro dipeptides NH2–Pro -X dipeptides

Pro–AMC Gly–Pro–AMC Z–Pro–Ala Z–Lys–Pro–AMC Lys–Pro–AMC Lys–Ala–AMC Gly–Pro Pro–Ala

a

Not plant.

Although sensitive to thiol protease inhibitors, it is also inhibited by DFP and an active site serine necessary for catalytic activity has been identified [59]. Proline iminopeptidase has been partially purified from castor bean endosperm and has a pH optimum of 7.5. It is present in the dry seed and increases during germination [63]. A proline iminopeptidase with a pH optimum of between 7.5 and 8.0 and an apparent molecular mass of 55 kDa, has been purified from apricot seeds [64]. It is probably a tetramer but, unlike the prokaryotic enzyme, is very poor at cleaving Pro– Pro bonds. Proline iminopeptidase has also been purified from wheat leaves, where it has a pH optimum of 7.4 [65]. All three plant enzymes were sensitive to sulfhydryl reagents, but not to PMSF or even DFP in the case of apricot seeds. In each case, activity was measured with Pro– b-naphthylamide, which does not distinguish between proline iminopeptidase and prolinase. Only the apricot enzyme was shown to cleave a tripeptide (Pro– Gly – Gly), while both wheat and apricot proline iminopeptidase had low Km values with Pro– b-naphthylamide (10 and 24 mM, respectively). Mikola and Mikola [60] reported the cleavage of the dipeptide Pro– Ala by a 5-day scutellar extract at pH 5.1, indicating either proline iminopeptidase or prolinase activity. Two genes encoding proline iminopeptidases have been found in Arabidopsis thaliana (Table 3), as well as ESTs from barley, wheat, maize, sorghum and rice.

The purified enzyme had an apparent molecular weight of 75 kDa, and a pI of 4.8, with maximal activity at pH 7.3 and resembled the mammalian enzyme in many respects [59]. It was also active towards Ala– X bonds, but the rate of hydrolysis was low compared with Pro – X and did not hydrolyze Z–Pro –b-naphthylamide. The best substrate, with a kcat/Km of 69 mM − 1 s − 1 was Z –Ala –Gly –Pro –b-naphthylamide, although Ala was preferred at P2. Substrate specificity data suggest the presence of at least five subsites (S3, S2, S1, S%1 and S%2), with high stereospecificity at S2, S1 and S%1. The natural substrates for mammalian prolyl oligopeptidases often have a positively charged residue (Arg, Lys, His) at P2 [59]. A serine protease with some homology to human prolyl oligopeptidase has been purified from soybean cell cultures [67]. It is probably an oligopeptidase B, and not a prolyl oligopeptidase, since it is unable to cleave after proline. ESTs from barley, wheat, rice, maize and sorghum have been reported and two Arabidopsis prolyl oligopeptidase genes are in the database

7.6. Prolyl oligopeptidase Prolyl oligopeptidase is a serine protease found in bacteria and mammals. It cleaves on the carboxyl side of proline residues in oligopeptides, but cannot cleave proteins. Prolyl oligopeptidase will not cleave Pro–Pro bonds, nor N-blocked peptides of the sequence Z-Pro– X [59]. Yoshimoto et al. [66] detected prolyl endopeptidase activity in many plants, including spinach, carrot, green onion, burdock, parsley and string beans, using the substrate Z– Gly – Pro – b-naphthylamide, and purified the enzyme from carrots. It was inhibited by DFP and r-chloromercuribenzoate but not by PMSF.

Fig. 6. Diagrammatic representation of the cleavage specificity of the known proline- specific peptidases. The metallopeptidases aminopeptidase P (AP) and prolidase cleave X – Pro bonds, while the others, which are serine peptidases, cleave Pro – X bonds. Prolinase and prolidase are strict dipeptidases. DPP II (dipeptidyl peptidase II) and DPP IV (dipeptidyl peptidase IV) remove a dipeptide from the N-terminus, whereas CPP (prolyl carboxy-peptidase) cleaves the Cterminal amino acid residue.

D.J. Simpson / Plant Science 161 (2001) 825–838

(Table 3), but it is difficult to be certain that they code for prolyl oligopeptidases.

7.7. Dipeptidyl peptidase IV Dipeptidyl peptidase IV (DPPIV) cleaves dipeptides from the N-terminus of peptides with three or more residues, with proline the preferred residue at P1, although alanine is also accepted [59]. It will not cleave Pro – Pro bonds. DPPIV is a glycoprotein, with a molecular weight of about 88 kDa in rats and mice and is classified as a serine protease based on inhibition by DFP. Rats deficient in this protease lose weight when fed on proline-rich protein, such as gliadin, indicating that this protease is important in the hydrolysis of prolamins in mammals [59]. DPP IV activity has also been reported in insects, yeast and plants. In plants, DPP IV activity has been reported in poppy [68,69] and gherkin seedlings [70] and in ginseng callus [71] and is suggested to play a role in the mobilization and/or utilization of storage proteins during germination. A single gene for encoding for Arabidopsis DPP IV has been identified (Table 3), and ESTs from barley, wheat, rice, rye and sorghum are known. A dipeptidyl peptidase IV has been purified from green barley malt and identified on the basis of inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kDa (85 kDa after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Its substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa – Pro –AMC (Table 5). The best substrates were Xaa = Lys and Arg, while the poorest were Xaa=Asp, Phe and Glu [72]. The Km values ranged from 0.071 to 8.9 mM, compared with values of 9– 130 mM reported for mammalian DPP IV’s. Barley DPP IV has almost no activity at pH 5, the pH of the endosperm, so it is likely that it is localized in the embryo or the scutellum. Thus, barley DPP IV may be involved in this later step of storage protein mobilization, releasing dipeptides, tripeptides and amino acids in the scutellum and/or embryo. The X –Pro dipeptide products of DPP IV cleavage are not substrates for barley malt carboxypeptidase I [47] but would be cleaved by a prolidase or an aminopeptidase P, with the release of free proline.

835

Table 7 Kinetic constants for substrate degradation by barley DPP IV Substrate

% Activity

Km (mM)

Lys–Pro–AMC Arg–Pro–AMC Tyr–Pro–AMC Thr–Pro–AMC Ile–Pro–AMC Ala–Pro–AMC Met–Pro–AMC Ser–Pro–AMC His–Pro–AMC Pro–Pro–AMC Leu–Pro–AMC Asn–Pro–AMC Gly–Pro–AMC Gln–Pro–AMC Glu–Pro–AMC Phe–Pro–AMC Asp–Pro–AMC Lys–Ala–AMC Z–Gly–Pro–AMC Z–Phe–Ala–AMC Pro–AMC

100.0 61.1 10.5 9.8 7.5 5.2 3.7 3.6 3.1 2.9 1.6 0.83 0.68 0.55 0.26 0.21 0.04 1.93 N.D. N.D. N.D.

0.071 0.090 0.73 0.93 0.70 0.34 2.17 1.26 1.03 1.10 1.09 3.24 6.88 1.38 2.98 0.65 8.88 3.74 N.D. N.D. N.D.

N.D., no cleavage detected. From [72].

the preferred substrates of DPP II are tripeptides, and substrates with more than 4 residues are not cleaved [59]. No DNA sequences for DPP II are listed in the public databases, although the amino acid sequence of the first 41 residues of pig DPP II is known [73].

7.9. Others Two other proline-specific peptidases are known from the literature, but the likelihood of their involvement in prolamin degradation seems remote. Carboxypeptidase P (EC 3.4.17.16) is probably a serine peptidase which cleaves after proline in the penultimate position, but will also accept Ala or Gly at P1 [74]. A prolyl tripeptidyl peptidase, which is an exopeptidase with a strict requirement for proline at P1, has been isolated from Porphyromonas ginigi6alis (AB008194, [75]). This is a member of the prolyl oligopeptidase family of serine proteases, with particular homology to DPP IV. No plant homologues for carboxypeptidase P or prolyl tripeptidyl peptidase are known.

7.8. Dipeptidyl peptidase II 8. Concluding remarks Dipeptidyl peptidase II (DPP II) is similar to DPP IV in catalyzing the cleavage of N-terminal dipeptides from substrates. DPP II can be distinguished from DPP IV on the basis of substrate specificity, since only DPP II can cleave Xaa–Pro – Pro and, while both peptidases accept the substitution of Pro by Ala at P1, the cleavage rate with DPP IV is much slower (Table 7). In addition,

In spite of the large number of endoproteases in green barley malt, cereal prolamins can be completely degraded into oligopeptides by two cysteine endoproteases of the papain family. They are unusual in having a low pH optimum in comparison to papain and they have probably adapted to the acidic (pH 5) condi-

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D.J. Simpson / Plant Science 161 (2001) 825–838

tions in the endosperm of germinating cereal grains. The acidification of the endosperm by the aleurone is likely to be important in making prolamins susceptible to the initial proteolytic cleavage events. It would be interesting to discover the molecular basis for the large differences in pH optima between papain (pH 6.8) and barley EP-B (pH 4.0). Although the latter is very efficient in cleaving sites outside of the central proline/ glutamine-rich repeat domain, it has a very low affinity for synthetic substrates based on the most frequently cleaved secondary site— PQQP. It is perhaps surprising that either EP-A or EP-B has not evolved a greater affinity for proline at P2, as found for the ginger rhizome cysteine endoproteases GP-I and GP-II [76]. A preference for proline at P2 is likely to be incompatible with the large hydrophobic residues found in the primary EP-A and EP-B cleavage sites. The role of other endoproteases found in germinating cereal grains, such as legumain and serine proteases remains to be elucidated using both recombinant prolamin and isolated protein bodies. The cleavage of total C hordein by EP-B produces many peptides, some of which are small enough (B5 amino acids) to be transported into the scutellum via a peptide transporter. Many of the large oligopeptides are further degraded by one or more of the carboxypeptidases present in the endosperm. However, the nature of the most common secondary cleavage site (PQ¡QP) results in many oligopeptides with a proline residue next to the amino- or carboxy-terminal residue. These are extremely poor substrates for the conventional carboxypeptidases and require proline-specific exopeptidases for their degradation. The most likely candidate, based on pH optimum, is prolyl carboxypeptidase, which has been detected and partially characterized in barley and wheat by Mikola and co-workers [44,45,60]). Purification and substrate characterization of this enzyme from germinating cereals should be a priority, particularly since the complete DNA sequence is known for six different prolyl carboxypeptidases from Arabidopsis, as well as barley and other cereals. The other proline-specific peptidases may have a role in general housekeeping in all cells, but are perhaps involved in the hydrolysis of the small proline-containing peptides transported from the endosperm. The role of dipeptidyl peptidase IV, prolidase, aminopeptidase P and prolyl iminopeptidase in the scutellum of germinating grains should be examined. Two cysteine endoproteases are involved in prolamin degradation in barley - EP-A and EP-B, which differ in sequence but are relatively similar in substrate specificity. Rice also has two such proteases (REP-A and REP-1) and a search of the EST database reveals two potential orthologs in sorghum (BE357460 and BE358196) and wheat (BF293987 and BF293204), so this may be true for all cereals. These proteases are

unusual in being secreted, presumably because they lack a vacuolar targeting sequence. If they have evolved from the so-called ‘KDEL’ cysteine endoproteases, then it is predicted that they would be secreted from the cell upon removal of the coding sequence for the C-terminal KDEL motif. Acknowledgements I thank my colleague Verena Cameron-Mills for helpful discussions and for assistance with the manuscript. I am grateful to Anne Davy, Nina Terp, Michael Blom Sørensen, Jacques Rouster and KarlKristian Thomsen for their valuable contributions. References [1] P.R. Shewry, M.J. Miles, A.S. Tatham, The prolamin storage proteins of wheat and related cereals, Prog. Biophys. Mol. Biol. 61 (1994) 37 – 59. [2] C.F. Higgins, J.W. Payne, Peptide transport by germinating barley embryos: evidence for a single common carrier for di- and oligopeptides, Planta 138 (1978) 217 – 222. [3] T. Sopanen, M. Uuskallio, S. Nyman, J. Mikola, Characteristics and development of leucine transport activity in the scutellum of germinating barley grain, Plant Physiol. 65 (1980) 249 –253. [4] T. Sopanen, E. Va¨ isia¨ nen, Uptake of glutamine by the scutellum of germinating barley grain, Plant. Physiol. 78 (1985) 684 –689. [5] E. Va¨ isia¨ nen, T. Sopanen, Uptake of proline by the scutellum of germinating barley grain, Plant. Physiol. 80 (1986) 902 –907. [6] N. Zhang, B.L. Jones, Characterization of germinated barley endoproteolytic enzymes by two-dimensional gel electrophoresis, J. Cer. Sci. 21 (1995) 145 – 153. [7] I. Schechter, A. Berger, On the size of the active site in proteases. I. Papain, Biochem. Biophys. Res. Commun. 27 (1967) 157 –162. [8] M. Meldal, K. Breddam, Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin, Anal. Biochem. 195 (1991) 141 –147. [9] E.D. Matayoshi, G.T. Wang, G.A. Krafft, J. Erickson, Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer, Science 247 (1990) 954 – 958. [10] A.D. Shutov, I.A. Vaintraub, Degradation of storage proteins in germinating seeds, Phytochemistry 26 (1987) 1557 – 1566. [11] W. Paul, J. Amiss, R. Try, U. Praekelt, R. Scott, H. Smith, Correct processing of the kiwifruit protease actinidin in transgenic tobacco requires the presence of the C-terminal propeptide, Plant Physiol. 108 (1995) 261 – 268. [12] M. Schmidt, D. Simpson, C. Gietl, Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endoprotease from ricinosomes, Proc. Natl. Acad. Sci. USA 96 (1999) 14159 – 14164. [13] T.-M. Enari, Proteinases and peptidases of malt and their influence on wort composition and beer quality, Cerevisia 1 (1986) 19 – 28. [14] E.G. deBarros, B.A. Larkins, Cloning of a cDNA encoding a putative cysteine protease from germinating maize seeds, Plant Sci. 99 (1994) 189 – 197. [15] A. Bottari, A. Capocchi, D. Fontanini, L. Galleschi, Major proteinase hydrolysing gliadin during wheat germination, Phytochemistry 43 (1996) 39 – 44.

D.J. Simpson / Plant Science 161 (2001) 825–838 [16] S.M. Koehler, T.-H.D. Ho, Purification and characterization of gibberellic acid-induced cysteine endoproteases in barley aleurone layers, Plant Physiol. 87 (1988) 95 –103. [17] S.M. Koehler, T.-H.D. Ho, A major gibberellic acid-induced barley aleurone cysteine proteinase which digests hordein, Plant Physiol. 94 (1990) 251 –258. [18] S.M. Koehler, T.H. Ho, Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers, Plant Cell 2 (1990) 769 – 783. [19] S. Marttila, I. Porali, T.-H.D. Ho, A. Mikkonen, Expression of the 30 kDa cysteine endoprotease B in germinating barley seeds, Cell Biol. Int. 17 (1993) 205 –212. [20] A. Davy, I. Svendsen, S.O. Sørensen, M.B. Sørensen, J. Rouster, M. Meldal, D.J. Simpson, V. Cameron-Mills, Substrate specificity of barley cysteine endoproteases EP-A and EP-B, Plant Physiol. 117 (1998) 255 –261. [21] L. Tamas, J. Greenfield, N.G. Halford, A.S. Tatham, P.R. Shewry, A beta-turn rich barley seed protein is correctly folded in Escherichia coli, Prot. Exp. Purif. 5 (1994) 357 –363. [22] B.L. Jones, M. Poulle, A proteinase from germinated barley II. Hydrolytic specificity of a 30 kilodalton cysteine proteinase from green malt, Plant Physiol. 94 (1990) 1062 –1070. [23] N. Zhang, B.L. Jones, Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley, Planta 199 (1996) 565 – 572. [24] A. Davy, M.B. Sørensen, I. Svendsen, V. Cameron-Mills, D.J. Simpson, Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B, based on the kinetics of synthetic peptide hydrolysis, Plant Physiol. 122 (2000) 137 – 145. [25] K. Kobrehel, J.H. Wong, A. Balogh, F. Kiss, B.C. Yee, B.B. Buchanan, Specific reduction of wheat storage proteins by thioredoxin h, Plant Physiol. 99 (1992) 919 –924. [26] J.H. Pheifer, D.E. Briggs, Thiols and disulphides in quiescent and germinating barley grains, both dormant and mature, J. Inst. Brew. 101 (1991) 85 –93. [27] E.G. deBarros, B.A. Larkins, Purification and characterization of zein-degrading proteases from endosperm of germinating maize seeds, Plant Physiol. 94 (1990) 297 –303. [28] K. Sutoh, H. Kato, T. Minamikawa, Identification and possible roles of three types of endopeptidase from germinated wheat seeds, J. Biochem. 126 (1999) 700 – 706. [29] H. Kato, T. Minamikawa, Identification of a rice cysteine endopeptidase that digests glutelin, Eur. J. Biochem. 239 (1996) 310 – 316. [30] N. Terp, K.K. Thomsen, I. Svendsen, A. Davy, D.J. Simpson, Purification and characterization of hordolisin, a subtilisin-like serine endoprotease from barley, J. Plant Physiol. 156 (2000) 468 – 476. [31] I. Besse, J.H. Wong, K. Kobrehel, B.B. Buchanan, Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium, Proc. Natl. Acad. Sci. USA 93 (1996) 3169 –3175. [32] M.A. Belozersky, S.T. Sarbakanova, Y.E. Dunaevsky, Aspartic proteinase from wheat seeds: isolation, properties and action of gliadin, Planta 177 (1989) 321 –326. [33] Y.E. Dunaevsky, S.T. Sarbakanova, M.A. Belozersky, Wheat seed carboxypeptidase and joint action on gliadin of proteases from dry and germinating seeds, J. Exp. Bot. 40 (1989) 1323 – 1329. [34] P. Runeberg-Roos, K. To¨ rma¨ kangas, A. O8 stman, Primary structure of a barley-grain aspartic proteinase. A plant aspartic proteinase resembling mammalian cathepsin D, Eur. J. Biochem. 202 (1991) 1021 – 1027. [35] N. Hiraiwa, M. Kondo, M. Nishimura, I. Hara-Nishimura, An aspartic endopeptidase is involved in the breakdown of propeptides of storage proteins in protein-storage vacuoles of plants, Eur. J. Biochem. 246 (1997) 133 –141.

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[36] K. To¨ rma¨ kangas, J. Kervinen, A. O8 stman, T. Teeri, Tissue-specific localization of aspartic proteinase in developing and germinating barley grains, Planta 195 (1994) 116 – 125. [37] S. Marttila, B.L. Jones, A. Mikkonen, Differential localization of two acid proteinases in germinating barley (Hordeum 6ulgare) seed, Physiol. Plant. 93 (1995) 317 – 327. [38] P. Sarkkinen, N. Kalkkinen, C. Tilgmann, J. Siuro, J. Kervinen, L. Mikola, Aspartic proteinase from barley grains is related to mammalian and lysosomal cathepsin D, Planta 186 (1992) 317 – 323. [39] J. Kervinen, P. Sarkkinen, N. Kalkkinen, L. Mikola, L.M. Saarma, Hydrolytic specificity of the barley grain aspartic proteinase, Phytochemistry 32 (1993) 799 – 803. [40] R. Wrobel, B.L. Jones, Identification and partial characterization of high Mr neutral proteinases from 4-day germinated barley seed, J. Cer. Sci. 18 (1993) 225 – 237. [41] R. Wrobel, B.L. Jones, Electrophoretic study of substrate and pH dependence of endoproteolytic enzymes in green malt, J. Inst. Brew. 98 (1992) 471 – 478. [42] M.A. Belozersky, Y.E. Dunaevsky, N.E. Voskoboynikova, Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds, Biochem. J. 272 (1990) 677 –682. [43] L. Kolehmainen, J. Mikola, Partial purification and enzymatic properties of an aminopeptidase from barley, Arch. Biochem. Biophys. 145 (1971) 632 – 642. [44] L. Mikola, Germinating barley grains contain five acid carboxypeptidases with complementary substrate specificities, Biochim. Biophys. Acta 747 (1983) 241 – 252. [45] L. Mikola, Acid carboxypeptidases in grains and leaves of wheat, Triticum aesti6um L., Plant Physiol. 81 (1986) 823 – 829. [46] K. Breddam, S.B. Sørensen, M. Ottesen, Isolation of carboxypeptidase II from malted barley by affinity chromatography, Carlsberg Res. Commun. 50 (1985) 199 – 209. [47] K. Breddam, S.B. Sørensen, Isolation of carboxypeptidase III from malted barley by affinity chromatography, Carlsberg Res. Commun. 52 (1987) 275 – 283. [48] K. Breddam, S.B. Sørensen, I. Svendsen, Primary structure and enzymatic properties of carboxypeptidase II from wheat bran, Carlsberg Res. Commun. 52 (1987) 297 –311. [49] S.B. Sørensen, K. Breddam, M. Ottesen, Primary structure of carboxypeptidase II from malted barley by affinity chromatography, Carlsberg Res. Commun. 52 (1987) 285 – 295. [50] F. Dal Degan, A. Rocher, V. Cameron-Mills, D. von Wettstein, The expression of serine carboxypeptidases during maturation and germination of the barley grain, Proc. Natl. Acad. Sci. USA 91 (1994) 8209 – 8213. [51] K. Olesen, M. Meldal, K. Breddam, Extended subsite characterization of carboxypeptidase Y using substrates based on intramolecularly quenched fluorescence, Prot. Pept. Lett. 3 (1996) 67 – 74. [52] J.C. Rogers, D. Dean, G.R. Heck, Aleurain: a barley thiol protease closely related to mammalian cathepsin H, Proc. Natl. Acad. Sci. USA 82 (1985) 6512 – 6516. [53] H. Watanabe, K. Abe, Y. Emori, H. Hosoyama, S. Arai, Molecular cloning and gibberellin-induced expression of multiple cysteine proteinases of rice seeds (oryzains), J. Biol. Chem. 266 (1991) 16897 – 16902. [54] C. Domoto, H. Watanabe, M. Abe, K. Abe, S. Arai, Isolation and characterization of two distinct cDNA clones encoding corn seed cysteine proteinases, Biochim. Biophys. Acta 1263 (1995) 241 – 244. [55] B.C. Holwerda, N.J. Galvin, T.J. Baranski, J.C. Rogers, In vitro processing of aleurain, a barley vacuolar thiol protease, Plant Cell 2 (1990) 1091 – 1106. [56] T. Sopanen, J. Mikola, Purification and partial characterization of barley leucine aminopeptidase, Plant Physiol. 55 (1975) 809 – 814.

838

D.J. Simpson / Plant Science 161 (2001) 825–838

[57] A. Mikkonen, Purification and characterization of leucine aminopeptidase from kidney bean cotyledons, Physiol. Plant. 84 (1992) 393 – 398. [58] T. Sopanen, Purification and partial characterization of dipeptidase from barley, Plant Physiol. 57 (1976) 867 – 871. [59] D.F. Cunningham, B. O’Connor, Proline specific peptidases, Biochim. Biophys. Acta 1343 (1997) 160 –186. [60] L. Mikola, J. Mikola, Mobilization of proline in the starchy endosperm of germinating barley grain, Planta 149 (1980) 149 – 154. [61] Y. Kubota, S. Shoji, K. Motohara, Purification and properties of prolidase from germinating soybeans, J. Pharm. Soc. Jpn. 97 (1977) 111 – 115. [62] K. Olesen, K. Breddam, Substrates with charged P1 residues are efficiently hydrolyzed by serine carboxypeptidases when S3-P1 interactions are facilitated, Biochemistry 36 (1997) 12235 – 12241. [63] R.E. Tully, H. Beevers, Proteases and peptidases of castor bean endosperm. Enzyme characterization and changes during germination, Plant Physiol. 62 (1978) 746 –750. [64] K. Ninomiya, K. Kawatani, S. Tanaka, S. Kawata, S. Makisumi, Purification and properties of a proline iminopeptidase from apricot seeds, J. Biochem. 92 (1982) 413 –421. [65] S.P. Waters, M.J. Dalling, Purification and characterization of an iminopeptidase from the primary leaf of wheat (Triticum aesti6um L.), Plant Physiol. 73 (1983) 1048 – 1054. [66] T. Yoshimoto, A.K.M. Adbus Sattar, W. Hirose, D. Tsuru, Studies of prolyl endopeptidase from carrot (Daucus carota): purification and enzymatic properties, Biochim. Biophys. Acta 916 (1987) 29 –37. [67] Z.-J. Guo, C. Lamb, R.A. Dixon, A serine protease from suspension-cultured soybean cells, Phytochemistry 47 (1998) 547 – 553.

[68] M. Benesˇova´ , P. Kova´ cs, M. Psˇena´ k, A. Barth, Dipeptidyl peptidase of poppy seedlings, Biologia 42 (1987) 779 – 787. [69] J. Stano, P. Kova´ cs, M. Psˇena´ k, J. Gajdosˇ, K. Erdelsky´ , D. Ka´ koniova´ , K. Neubert, Distribution of dipeptidyl peptidase IV in organ tissue cultures of poppy plants Papa6er somniferum L. cv. ‘Amarin’, Pharmazie 52 (1997) 319 – 321. [70] J. Stano, P. Kova´ cs, P. Nemec, K. Neubert, Dipeptidyl peptidase IV in gherkin seedlings Cucumis sati6us L. cv. Pa´ lava, Biologia 49 (1994) 905 – 910. [71] J. Stano, P. Kova´ cs, D. Ka´ koniova´ , D. Lisˇkova´ , N.D. Kirilova, V.P. Komov, Activity of dipeptidyl peptidase IV in ginseng callus culture, Biologia 49 (1994) 353 – 357. [72] A. Davy, K.K. Thomsen, M. Juliano, L.C. Alves, I. Svendsen, D.J. Simpson, Purification and characterization of a barley dipeptidyl peptidase IV, Plant Physiol. 122 (2000) 425 –431. [73] K. Huang, M. Takagaki, K. Kani, I. Ohkubo, Dipeptidyl peptidase II from porcine seminal plasma, purification, characterization, and its homology to granzymes, cytotoxic cell proteinases (CCP 1-4), Biochim. Biophys. Acta 1290 (1996) 149 – 156. [74] S. Hedeager-Sørensen, A.J. Kenny, Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys, Biochem. J. 229 (1985) 251 – 257. [75] A. Banbula, P. Mak, M. Bugno, J. Silberring, A. Dubin, D. Nelson, J. Travis, J. Potempa, Prolyl tripeptidyl peptidase from Porphyromonas gingi6alis. A novel enzyme with possible pathological implications for the development of periodontitis, J. Biol. Chem. 274 (1999) 9246 – 9252. [76] K.H. Choi, R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516 – 1526.