wheat inhibitors of heterologous a-amylases

Plant Physiol. (1991) 96, 768-7740032-0889/91 /96/0768/07/$01 .00/0

Received for publication September 14, 1990Accepted January 23, 1991

Wheat Inhibitors of Heterologous a-Amylases'

Characterization of Major Components from the Monomeric Class

Luis Gomez, Rosa Sanchez-Monge, Carlos Lopez-Otin, and Gabriel Salcedo*

Departamento de Bioquimica, E. T.S. Ingenieros Agr6nomos, 28040 Madrid, Spain (L.G., R.S.-M., G.S.); andDepartamento de Bioquimica, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain (C.L. -0.)

ABSTRACT

The four major components of the wheat monomeric a-amylaseinhibitors (WMAI) from wheat, Triticum aestivum, endosperm havebeen isolated and characterized. Two of them, WMAI-1 andWMAI-2, are highly active against the a-amylase from the insectTenebrio monitor and their N-terminal amino acid sequences in-dicate that they are closely related to each other (86% identicalresidues) and to the other members of the family (subunits ofdimeric and tetrameric a-amylase inhibitors and trypsin inhibi-tors). WMAI-1, which is identical to the previously described 0.28inhibitor, is encoded by a gene located in the short arm ofchromosome 6D and WMAI-2 by a gene in the short arm ofchromosome 6B. Components 3 and 4, which have blocked N-terminal residues, have identical internal amino acid sequencesand are a separate class of proteins with respect to WMAI-1 andWMAI-2, although their amino acid composition and apparentmolecular weights are quite similar. Their inhibitory activity versusa-amylases is either unstable during the purification process ordue to contamination with other inhibitors.

some detail (2, 4, 11, 17, 22, 24, 25), only one member of themonomeric class, designated 0.28, has been characterized(19). As this wheat inhibitor type seems to be the most activeagainst the a-amylases of phytophagus insects (8, 12), furtherstudy of the in vitro properties and genetic control of othermembers ofthe monomeric class is warranted. We now reportsuch a study for a homologous variant of 0.28 and furthershow that two other proteins of similar size and solubilityproperties belong to a different protein family.

MATERIALS AND METHODS

Plant Material

Ground endosperms from Triticum aestivum cv ChineseSpring and Triticum turgidum cv Senatore Capelli were usedas starting material for the isolation of inhibitors. Compen-sated nulli-tetrasomic and ditelosomic lines of T. aestivum cvChinese Spring (gift of E. R. Sears, Columbia, MO) were usedto locate controlling genes for the inhibitors.

Proteinaceous inhibitors of a-amylases and proteases are

quite abundant in cereal endosperm, where a single inhibitorfamily represents a substantial fraction of the albumins andglobulins (see refs. 8, 12 for reviews). This protein family,which has been particularly studied in wheat and barley,includes the 12 to 15 kD subunits of tetrameric, dimeric, andmonomeric inhibitors ofheterologous a-amylases and oftryp-sin (8, 12). A role in plant protection has been postulated forthese inhibitors, based on their activity against animal a-

amylases and lack of it against plant enzymes, as well as on

their effects on insect development (1, 12, 13). Furthermore,several members of this family are major allergens in baker'sasthma, the main allergy associated with flour manipulation(3, 15). There is some evidence that two wheat componentsmight be relevant in connection with the surface propertiesof pasta products ( 14, 20).Although the polimorphism and genetic control of subunits

from the dimeric and tetrameric classes have been studied in

Supported by Comision Interministerial de Ciencia y Tecnologia(grant AL89/0 121).

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Inhibitors Purification

The crude inhibitor preparations from both wheat specieswere obtained by 0.15 M NaCl extraction and (NH4)2SO4precipitation as previously described (16). These preparationswere fractionated by gel filtration on Sephadex G-100 aspreviously described (16). Fractions around 12.5 kD with highactivity against Tenebrio molitor a-amylase (monomeric in-hibitors) were pooled, lyophilized, and chromatographed ona preparative RP-HPLC2 column (Vydac-C4, 22 mm x 250mm, particle size 10 ,um), using a two-step linear gradient 20to 50% (2 mL/min; linear 20-35% gradient in 140 min, linear35-50% gradient in 100 min) of acetonitrile in 0.1% trifluo-roacetic acid. Fractions containing inhibitors were lyophilizedand rechromatographed on a semipreparative RP-HPLC Ul-trapore-C3 column (10 x 250 mm, particle size 5 gm), andeluted with a linear gradient 15 to 35% of isopropanol in0.1% TFA (0.5 mL/min).

2 Abbreviations: RP-HPLC, reverse phase-high performance liquidchromatography; IEF, isoelectrofocusing; SGE, starch gel electropho-resis; WMAI, wheat monomeric a-amylase inhibitor.

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WHEAT MONOMERIC a-AMYLASE INHIBITORS

IIEF pH8-o

IN

wC,Co

Protein concentration was determined by the methods ofLowry et al. (23) and Smith et al. (30).

Electrophoretic Procedures

SDS-PAGE was performed according to Laemmli (21).Two dimensional electrophoresis (IEF x SGE) was carriedout as previously described ( 16).

Amino Acid Analysis and Protein Sequencing

Protein hydrolysis at 24, 48, and 72 h was carried outaccording to Moore and Stein (26), and performic acid oxi-dation as in Hirs (18). Amino acid analyses were performedfollowing Bidlingmeyer et al. (6), on an HPLC BeckmanSystem.Enzymatic hydrolysis of purified proteins was performed

by endoproteinase Lys-C (EC 3.4.99.30), during 18 h at 370C,using a 25 mM Tris-HCl (pH 8.5), 1 mm EDTA buffer. Theresulting peptides were fractionated by RP-HPLC on a semi-preparative Nucleosil 300-5 C4 column (8 x 250 mm, particlesize 5 pm), using a linear gradient (0-70%) of acetonitrile in0.1% TFA over a period of 70 min (1 mL/min, previouselution with 0.1% TFA during 5 min).

Protein sequencing was performed by standard methods,using an Applied Biosystems 470A gas phase sequenator.

-Jcc

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Analysis of Genetic Stocks

Individual kernels of the compensated nulli-tetrasomic andditelosomic lines of T. aestivum cv Chinese Spring weredelipidated with petroleum ether (boiling point 50-700C, 1 x

kD

TIME (min)

Figure 1. A, B, Two-dimensional electrophoretic maps (IEF x SGE)of the crude inhibitor preparation (A) and of the gel-filtration fractioncorresponding to monomeric inhibitors (B) from T. aestivum. C, RP-HPLC fractionation of the 12.5 kD gel-filtration fraction (monomericinhibitors). Positions of the major components 1 to 4 are indicated.

29 -

21 -

12.5-

Inhibition Tests

Inhibitory activity against Tenebrio monitor or human salivaa-amylases (EC 3.2.1.1) was tested by the method of Bernfeld(5), with minor modifications previously described (16). Allassays were carried out using 1 unit of a-amylase, defined as

the amount of enzyme required to produce the reducingequivalents of 1 gmol of maltose in our experimentalconditions.

Trypsin (EC 3.4.2.1.4) inhibition was tested as in Boisenand Djurtoft (7).

6.5-

I I I 1 1

4 3 2 1 F T

Figure 2. SDS-PAGE of the following samples: T, crude inhibitorpreparation from T. aestivum; F, 12.5 kD gel-filtration fraction (mon-omeric inhibitors); 1 to 4, purified components 1 to 4, respectively.The molecular masses (in kD) of reference proteins appear on the leftside of the figure.

A1 2

* 0

B1 2U *

4 30

1.5-

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Plant Physiol. Vol. 96, 1991

100- higher than that of the 0.19 dimeric inhibitor (25) used asCOMP. I control (Fig. 3). No inhibition was detected for purified pro-

teins 3 and 4 (using up to 10 gg of protein/assay), although80- significant activities were found in the RP-HPLC peaks cor-responding to these two components in Figure IC. None of

/compMP. 2 T the purified proteins were active against human salivary a-NO-1 I/ / _ amylase or against trypsin, except component 1, which was

60-WDAI - I ~~~-weakly active against the human a-amylase (about 10-fold2 less active than against the insect one).E... I 11 /Endosperm from tetraploid wheat (T. turgidum cv Senatorem I ,' I / Capelli) was subjected to the same extraction and fractiona-T 40 - tion procedure followed for hexaploid wheat. In this case,

only two main components were detected in the monomeric,COMP. 3t inhibitors preparation (Fig. 4A), which comigrated with pro-

20- 1l to of teins 3 and 4 of T. aestivum when separated by SDS-PAGE,by two-dimensional electrophoresis, or by RP-HPLC. Toclarify their putative inhibitory activities, both componentswere isolated from tetraploid wheat. The two proteins were

0- homogeneous after the first RP-HPLC step (Fig. 4B), and0 1 2 5 only component 3 was significantly active against the a-

PROTEIN (Mg) amylase of T. monitor (Fig. 3). When purified component 3

Figure 3. Inhibitory activities against T. monitor a-amylase of purifiedcomponents 1 and 2 from T. aestivum and component 3 from T.turgidum (comp. 3J). WDAI-1 (0.19 wheat dimeric inhibitor) was usedas control. Inhibition assays were carried out using 1 unit a-amylase Aas defined in "Materials and Methods." Vertical bars correspond tostandard deviations.

10 w/v, 1 h). The residues were extracted twice with 70%(v/v) ethanol (1 x 10 w/v, 1 h), and the extracts werefractionated by two-dimensional electrophoresis (IEF x SGE)as mentioned above.

RESULTS

Isolation of Major Components of MonomericInhibitors from T. aestivum and T. turgidumA fraction enriched in monomeric inhibitors of heterolo-

gous a-amylases was obtained from hexaploid wheat (T.aestivum cv Chinese Spring) by salt-extraction, (NH4)2SO4-precipitation, and gel filtration on Sephadex G-100 as previ-ously described (16; not shown). This fraction, which had anapparent Mr of about 12,500 and was highly active againstthe a-amylase of the insect T. monitor, was found to containfour main components when analysed by two-dimensionalelectrophoresis (Fig. 1, A and B). The crude preparation ofmonomeric inhibitors was subjected to RP-HPLC using anH20-acetonitrile gradient (Fig. IC), and only those peaksincluding any of the four major components showed inhibi-tory activity. These proteins were obtained in homogeneousform by a further step of RP-HPLC using a H20-isopropanolgradient. Homogeneity was checked by SDS-PAGE (Fig. 2)and by two-dimensional electrophoresis (not shown).Only proteins 1 and 2 were found to be inhibitory after the

second RP-HPLC step. Their activities against the a-amylaseof T. monitor were very similar to each other and significantly

770 GOMEZ ET AL.

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Table I. Amino Acid Composition of Components 1 to 4 Isolated from T. aestivum (mol/1 00 mol ofamino acids)

The number of residues/molecule, adjusted to the size of component 1 (0.28 inhibitor; ref. 19), isshown in parentheses.

Amino Acida Component 1 Component 2 Component 3 Component 4

Lys 5.5 (7) 3.5 (4) 3.4 (4) 3.2 (4)His 0.4 (0) 1.8 (2) 2.3 (3) 2.6 (3)Arg 6.1 (7) 5.9(7) 5.0 (6) 3.8(5)Asx 7.4 (9) 6.9(8) 11.5 (14) 12.1 (14)Thr 3.1 (4) 5.0(6) 7.1 (8) 7.9(9)Ser 7.2 (9) 7.3 (9) 5.5 (6) 5.4 (6)Glx 10.5 (12) 10.7 (13) 9.2(11) 9.1 (11)Pro 7.3 (9) 7.1 (9) 5.0 (6) 5.0 (6)Gly 10.0 (12) 10.3 (12) 11.8 (14) 11.6 (14)Ala 8.4 (10) 7.8 (9) 11.9 (14) 12.0 (14)Val 11.3 (13) 9.7 (12) 6.9 (8) 6.8 (8)Cys 6.6 (8) 6.1 (7) 3.6 (4) 3.1 (4)Met 2.2 (3) 3.2 (4) 0.5 (1) 0.5 (1)lie 2.3 (3) 3.4 (4) 3.4 (4) 3.5 (4)Leu 7.8(9) 6.9(8) 5.7 (7) 5.5(7)Tyr 3.6(4) 2.7(3) 4.9 (6) 5.4(6)Phe 0.3 (0) 1.7 (2) 2.3 (3) 2.5 (3)

a Trp was not determined.

was subjected to a second RP-HPLC step, using the sameH20-isopropanol gradient which allowed its repurificationfrom hexaploid wheat, no inhibition was obtained against theinsect enzyme.

Amino Acid Composition and Protein Sequences ofPurified Components

The amino acid compositions of the four purified compo-nents were quite similar (Table I), but the indexes of relativecompositional differences in Table II clearly indicated thatthey could be grouped into two pairs: 1/2 and 3/4. Both theamino acid composition of component 1 and its position inthe two-dimensional protein maps suggested its identity withthe monomeric inhibitor 0.28 previously characterized (19,29). This fact was confirmed by coelectrophoresis of compo-nent 1 with an authentic sample of the inhibitor.The N-terminal sequence of component 2 was determined

and found to be homologous (86% identical residues) to thatpreviously reported for 0.28 (Fig. 5). We propose the desig-nations WMAI-1 and WMAI-2 for components 1 (0.28) and2, respectively.

Table II. Values of the Relative Compositional Difference Indexesfor the Binary Comparisons of Components 1 to 4

The indexes have been determined according to Cornish-Bowden(10), as modified by Paz-Ares et al. (27), using the data from Table I.

Component 2 3 4

1 0 1.62 1.742 1.38 1.463 L1.02

The N-terminal residues ofcomponents 3 and 4 were foundto be blocked. Enzymatic digestion of the purified compo-nents with endoproteinase Lys-C, followed by isolation andN-terminal sequencing of the major fragments obtained (Fig.6), fully confirmed that the two components were closelyrelated to each other: while peptide C seemed to have ablocked N-terminal residue in both cases, the sequences ofpeptides A and B were identical in both proteins. No signifi-cant homology was found between these sequences and anyof the known sequences of the different members of the a-

amylase/trypsin inhibitor family, or with any sequence fromavailable data banks.

Chromosomal Location of Genes EncodingMonomeric Inhibitors

Individual kernels of the nulli-tetrasomic and ditelosomicseries of lines from T. aestivum cv Chinese Spring wereextracted with 70% (v/v) ethanol. These extracts, which havebeen previously shown to contain subunits of the differentinhibitors (29), were subjected to two-dimensional electropho-resis. The spots corresponding to WMAI-l and WMAI-2 were

identified by coelectrophoresis with the pure proteins. Asshown in Figure 7, the two inhibitors were present in all linestested except those lacking the short arms of chromosomes6B (WMAI-2 absent) and 6D (WMAI-l absent).

PROTEIN 1 10 20 30

COMP. 2 S G P AIM GD P A rR[P T G C R A M V K L Q C V G S Q V P E ACOMP.1 (0.28) IS G P AS

cN P T G K L A T G C R A M V K L Q C V G S Q V P E A

Figure 5. Alignment of the N-terminal sequence of component 2 withthat of 0.28 inhibitor (component 1). The sequence of 0.28 was takenfrom Kashlan and Richardson (19).

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BPEPTIDE

A (COMP.

A (COMP.

-J

I-

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0

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TIME (min)

1

3)

4 )

B (COMP.

B (COMP.-

1 0

Y G R T A F C G P A

I D T N G I G Y Q Q G G L N V

Figure 6. A, RP-HPLC fractionation of the peptides obtained afterenzymatic hydrolisis of component 3 with endoproteinase Lys-C.Identical results were obtained in the case of component 4. PeptidesA, B, and C were subjected to N-terminal sequencing. B, N-terminalsequences of peptides A and B. Results were identical for compo-nents 3 and 4 in both peptides. Peptide C of Figure 6A had a blockedN-terminal residue.

N3W1" t.,

The lack of detection of components 3 and 4 in H20, 0.5M NaCl or 70% ethanol extracts did not allow for the chro-mosomal location of their encoding genes by the method usedin the case of WMAI- I and WMAI-2. Fractionation by othertwo-dimensional electrophoretic procedures (IEF x SDS-PAGE; IEF x PAGE), even followed by silver staining, wasalso unsuccessful.

DISCUSSION

The four major components of the monomeric inhibitorsfrom T. aestivum have been purified and characterized. Al-though their molecular size and amino acid composition are

quite similar, both the relative compositional difference in-dexes (Table II) and their inhibitory activities, indicate thattwo different pairs of closely related components can bedistinguished: WMAI- l/WMAI-2 and components 3/4.WMAI- and WMAI-2 are highly active against T. monitor

a-amylase. The sequence homology between these two inhib-itors and the equivalent positions of their respective genes inthe short arms of chromosomes 6D and 6B clearly show thatthe two genes are homoeologous. These results are in linewith those described for the subunits oftetrameric and dimerica-amylase inhibitors, which are also encoded by pairs ofhomoeologous genes located in the short arms ofchromosomegroups 3, 4, and 7 of the B and D genomes ofhexaploid wheat(12). No monomeric inhibitor has been found in associationwith the A genome, as is the case for the other inhibitorclasses. However, DNA sequences hybridizing with clonedDNA corresponding to the subunits of tetrameric inhibitorswere detected in the predictable A genome chromosomes(11). In the present case, a cDNA probe corresponding toWMAI-1 (0.28 inhibitor) does hybridize with DNA in theshort arms of chromosomes 6B and 6D, but does not recog-nize any DNA fragment from the A genome (M. Mena, P.Carbonero, unpublished data). Although the gene encodingWMAI-2 is located in the B genome, the inhibitor seems tobe absent in T. turgidum cv Senatore Capelli (genomesAABB). This inhibitor probably corresponds in the two-di-mensional protein maps published by Rodriguez-Loperena et

t-, H tj *

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al. (28) to spot No. 2, which was absent in most hexaploidand tetraploid wheats analyzed, including cv Senatore Capelli.

In relation to proteins 3 and 4, the other pair of majorcomponents present in the monomeric inhibitors preparation,it can be concluded that they are not homologous to the aboveinhibitors, and therefore do not belong to the same proteinfamily. Recently, Carrano et al. (9) have isolated from hex-aploid wheat a putative variant of the monomeric inhibitors(protein 0.14) which is inactive against heterologous a-amy-lases. The apparent Mr and amino acid composition of thisprotein are quite close to those ofcomponents 3 and 4 (relativecompositional divergence indexes are 0.32 and 0.34, respec-tively, values that are within those expected for separatepurifications of the same protein) and significantly differentfrom those of the monomeric inhibitors. The results presentedhere indicate that proteins 3 and 4 are either very closelyrelated to each other or different modifications of the sameprotein. The fact that the diploid species T. tauschii (genomesDD) shows only one of these components, corresponding toprotein 3 of T. aestivum (L. Gomez, R. Sanchez-Monge, G.Salcedo, unpublished data), suggests that indeed they mightbe two homeologous proteins. Whether or not they are trueinhibitors of heterologous a-amylases remains open becauseour observations allow for two alternative explanations: theinhibitory activity of RP-HPLC fractions corresponding toproteins 3 and 4 might be due to contamination with otherinhibitors, which could be eliminated in the second RP-HPLCstep, or the loss of activity in this step could be due todenaturation. The results obtained in the case of T. turgidum,which lacks WMAI-1 and WMAI-2, suggest that this newclass of proteins could be also inhibitors of heterologous a-amylases, albeit less stable during purification than the pre-viously described family.The possible relevance of the inhibitors in plant defense is

currently being tested by transgenically expressing gene con-structions encoding inhibitor WMAI- 1 and challenging withpertinent pest (P. Carbonero, unpublished data).

ACKNOWLEDGMENTS

We thank F. Garcia-Olmedo for discussion, encouragement andrevision of the manuscript, and J. Garcia-Guijarro and D. Lamonedafor technical assistance.

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