Botanical Studies (2009) 50: 149-158.

7

These two authors contributed equally to this works.

*

Corresponding author: E-mail: boyhlin@gate.sinica.edu.tw;

Fax: +886-2-2782-7954; Tel: +886-2-2787-1172 (Yaw-Huei

LIN); E-mail: gjhuang@mail.cmu.edu.tw (Guan-Jhong

HUANG).

INTRODUCTION

Aspartic proteinases (APs) (aspartic endopeptidases,

EC, 3.4.23) are one of the four main classes of proteinases,

the others being serine, cysteine, and metallo-proteinases

(Barrett, 1998) and are a widely distributed class of

proteinases present in animals, microbes, viruses, and

plants (Davies, 1990; Rawling and Barret, 1995; Mutlu

and Gal, 1999; Simoes and Faro, 2004). Plant APs have

characteristics common with aspartic proteinase A1

family, are active at acidic pH, are specifically inhibited by

pepstatin and have two aspartic acid residues responsible

for the catalytic activity (Dunn, 2002). APs have been

Cloning and expression of aspartic proteinase cDNA

from sweet potato storage roots

Guan-Jhong HUANG

1,7

, Shyh-Shyun HUANG

1,7

, Hsien-Jung CHEN

2

, Yuan-Shiun CHANG

1

, Shu-

Jen CHANG

3

, Heng-Yuan CHANG

1

, Po-Chow HSIEH

1

, Man-Jau CHANG

4

, Ying-Chih LIN

5

, and

Yaw-Huei LIN

6,

*

1

Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung 404, Taiwan

2

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan

3

School of Pharmacy, China Medical University, Taichung 404, Taiwan

4

Deparment of Applied Cosmetics Science, Ching Kuo Institute of Managment and Health, Kee-Lung 203, Taiwan

5

Nursing and Management, Jen-Teh Junior College of Medicine, Mioali 356, Taiwan

6

Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan

(Received June 13, 2008; Accepted November 27, 2008)

ABSTRACT.

Aspartic proteinases (EC, 3.4.23) cDNA clone (SPAP) of sweet potato (Ipomoea batatas

(L.) Lam. ¡¥Tainong 57¡¦) storage roots were isolated by differential display. The open reading frame in this

cDNA encodes a pre-pro-protein of 508 amino acids with a predicted molecular mass of 55,006 Da (pI 4.91).

The SPAP gene shares 81% and 78% homology on the level of nucleotides and amino acids, respectively,

with an aspartic proteinase cDNA of sweet potato senescent leaves (SPAPSL). SPAP amino acid sequence

was different from other AP sequences in signal and propeptide portions. The deduced amino acid sequence

contains the conserved features of plant aspartic proteinases, including the plant specific insert (PSI) and two

active site aspartic acid residues. Examination of the expression patterns in sweet potato by northern blot

analyses revealed that the transcripts of SPAP were specifically induced in the storage roots. Recombinant

SPAP overproduced in E. coli (M15) was purified by Ni

2+

-chelated affinity chromatography. Active

recombinant SPAP was able to digest the 22 kDa sweet potato trypsin inhibitor (TI) when the latter was

reduced by dithiothreitol (DTT). SPAP could not degrade bands of reduced TI when NTS (NADP/thioredoxin

system) was used to reduce TI. These results suggest that SWAP has an in vivo proteolytic function of

processing storage SPTI after its being degraded initially by a specific cysteine proteinase.

Keywords: Aspartic proteinase; cDNA sequence; Recombinant protein; Sweet potato; Trypsin inhibitor.

found in seeds, tubers, flowers, and petals of many

species. A number of aspartic proteinases cDNAs have

been isolated from different plants including Arabidopsis,

Brassica, rice, barley, and tomato (Runeberg-Roos et al.,

1991; Asakura et al., 1995; Schaller et al., 1996; D¡¦Hondt

et al., 1997; Hiraiwa et al., 1997; Xia et al., 2004). The

typical plant AP sequences contain preproportions which

are similar to those of the other species. Plant AP genes

have an extra region of approximately 100 amino acids

called as "plant specific insert" (PSI). This segment,

inserted into the C-terminal domain of the plant APs

precursors, is usually removed during the proteolytic

maturation of the proteinases. The PSI sequence shows

no homology with mammalian or microbial APs, but is

highly similar to that of saposin-like proteins (SAPLIPs)

(Guruprasad et al., 1994). PSI has been reported to

function as signals both for transport of AP molecules from

the endoplasmic reticulum (ER) and for their targeting to

the vacuole (Terauchi et al., 2006).

mOleCUlAR BIOlOgy

150

Botanical Studies, Vol. 50, 2009

APs were shown to perform many different and diverse

biological functions, including specific protein processing

(e.g. rennin and cathepsin D), protein degradation (e.g.

chymosin and pepsin) or viral polyprotein processing

(human immunodeficiency virus AP) (Rawlings et al.,

1995; Hiraiwa et al., 1997; Mutlu and Gal, 1999). Plant

AP functions are predicted in studies of the processing or

degradation of putative protein substrates in vitro and/or

specific expression in certain tissues or under specific

conditions. Plant APs have been implicated in protein

processing and/or degradation in different plant organs, as

well as in plant senescence, stress responses, programmed

cell death and reproduction (Simoes and Faro, 2004).

In this paper we report the isolation and characterization

of a sweet potato cDNA encoding AP. Active recombinant

SPAP protein was able to digest the reduced 22 kDa

trypsin inhibitor protein, one of the storage proteins of

sweet potato tuberous roots.

mATeRIAlS AND meTHODS

Plant materials

Fresh storage roots of sweet potato (Ipomoea batatas

(L.) Lam. ¡¥Tainong 57¡¦) were purchased from a local

market. After cleaning with water, the roots were placed in

a thermostated (28oC) growth chamber and sprayed with

water twice a day. Sprouted plants were cultivated in the

greenhouse to collect roots, stems and full expanded green

leaves for experiments.

PCR-based subtractive hybridization and RACe

PCR

Total RNA was isolated separately from the storage

roots and sprouts of roots of sweet potato according to the

method of Sambrook et al. (Sambrook et al., 1989). Then,

mRNA was purified with a purification kit (Promega) and

used for the differentially-expressed first strand cDNA

synthesis using a PCR-based subtractive hybridization

kit (Clontech) following the protocol supplied by the

manufacturer. The double-strand cDNAs of the storage

roots were subtracted by the sprouts of roots, then ligated

to the pGEM-T easy vector for E. coli DH5£\ competent

cell transformation. Recombinant plasmids were isolated

for DNA sequencing using the ABI PRIZM 337 DNA

Sequencer. Nucleotide sequence data were analyzed using

the Genetics Computer Group (GCG) programs. Full-

length cDNA clone was obtained by performing 5¡¦ and

3¡¦ RACE (5¡¦ and 3¡¦ rapid amplification of cDNA ends)

using the Marathon cDNA amplification kit (Clontech)

according to the manufacturer¡¦s instructions. The gene-

specific primers (5¡¬-TCTCC AGATA GGTTT TGCTG

AAGCT GCATG-3¡¬) were used to amplify the double

strand cDNAs.

expression of aspartic proteinase in E. coli

Its prosequence was expressed in E. coli. The coding

sequence was amplified from cDNA SPAP using an

oligonucleotide (5¡¦-GGA AA CCTTT GAGCA TGCC A

TGGAA ATATC-3¡¦), with a SphI site (underlined) at the

putative initial Met residue, and an oligonucleotide (5¡¦-

TCTCC AGATA GGTTT TGCGG TACC-3¡¦), with a KpnI

site at the 3¡¦ end. The PCR fragment was subcloned in

pGEM T-easy vector. The plasmid was then digested with

SphI and KpnI and the excised fragments were subcloned

in pQE30 expression vector (QIAexpress expression

system, Qiagen). The resulting plasmid, termed pQE-

SPAP, was introduced into E. coli (M15). Cultures of the

transformed E. coli (M15) overexpressed a protein of the

expected molecular mass, which was purified by affinity

chromatography in Ni-nitrilotriacetic acid (NTA) columns

(Qiagen), according to Huang et al (Huang et al., 2007).

DNA isolation and Southern blot analysis

Young leaves of sweet potato were harvested and

ground in liquid N

2

. The powder was transferred to a

centrifuge tube, mixed gently and thoroughly with CTAB

(cetyltrimethylammonium bromide) buffer (2% CTAB, 1.4

M NaCl, 20 mM EDTA, 2% 2-mercaptoethanol, and 100

mM Tris-HCl pH 8.0), and kept at 60¢XC in a water bath

for genomic DNA extraction according to the method of

Huang et al. (Huang et al., 2004a). The total nucleic acid

after precipitation with an equal volume of isopropanol

was re-dissolved in sterile water, digested with various

restriction enzymes and separated on 0.8% agarose gels.

After electrophoresis, the DNA was transferred onto a

Hybond-N

¡Ï

nylon membrane (Amersham) following the

protocol of Molecular Cloning (Sambrook et al., 1989) for

Southern blot hybridization.

RNA isolation and northern blot analysis

Total RNA was extracted from different tissues of sweet

potato with TRIzol reagents kit (Invitrogen) according

to the manufacturer¡¦s instructions. For northern blotting,

10 £gg of total RNA isolated from storage roots, sprouts,

sprouted roots, veins, fully expanded green leaves, and

flowers were applied to a formaldehyde denaturing

gel, then transferred to an Amersham Hybond-N

¡Ï

nylon membrane after electrophoresis, according to

the methods of Huang et al. (Huang et al., 2005a). The

filter was hybridized sequentially with £\-

32

P-labelled

AP full-length cDNA. The procedures for hybridization

and autoradiography were according to the methods of

Molecular Cloning (Sambrook et al., 1989). Visualization

of hybridization bands was carried out using X-ray film

(Kodak).

Purification of sweet potato trypsin inhibitor

Sweet potato storage roots were washed and peeled, and

then cut into strips that were extracted immediately and

processed according to Huang et al. (Huang et al., 2004b;

2008a). The crude extracts were loaded directly onto a

trypsin Sepharose-4B affinity column. The adsorbed TI

was eluted by pH changes with 0.2 M KCl (pH 2.0).

HUANG et al. ¡X Aspartic proteinase: cloning and expression

151

Protein staining and thiol-label staining of

trypsin inhibitors on 10% or 15% denaturing

polyacrylamide gels

Samples were mixed with sample buffer, namely 60

mM Tris-HCl buffer (pH 6.8) containing 2% SDS, 25%

glycerol, and 0.1% bromophenol blue with or without

2-mercaptoethanol. Coomassie brilliant blue G-250

was used for protein staining (Huang et al., 2008b). The

method of thiol-label staining on an SDS-PAGE gel

basically followed the report of Huang et al. (Huang et al.,

2005b) using the mBBr (monobromobimane) reagent as a

probe.

Production of polyclonal antibody and western

blot hybridization

Expressed SPAP protein was cut from the 15%

polyacylamide gel, and eluted with appropriate amount

of pH 7.5 phosphate buffer saline (PBS) containing 0.1%

SDS. The eluted proteins were precipitated with acetone

containing 10% trichloroacetic acid (TCA) at -20¢XC

for 2 h. After centrifugation at 13,000 g for 20 min, the

pellet was washed with acetone twice, then, dried at room

temperature. The acetone powder was re-dissolved in a

small amount of PBS containing 0.1% SDS and used as

antigens for subcutaneous injections of rabbit to prepare

the first antigens (Taiwan Bio-Pharm Inc.). The second

antigen (goat against rabbit Fc portion of Ig) was a

product of Sigma (USA). Polyclonal antibodies obtained

from rabbit antiserum were utilized for western blot

hybridization to study the gene expression of SPAP in

different tissues of sweet potato.

Protein extraction, electroblotting analysis of

aspartic proteinase and TI

All steps were carried out at 4-8¢XC. Sweet potato leaves,

sprouted storage roots, veins, and storage roots, were

cleaned and homogenized with 4 volumes (v/w) of 50

mM Tris-HCl buffer (pH 7.5) in a Polytron homogenizer

(Luzern, Swiss). The homogenate was filtered through

two layers of cheesecloth and then centrifuged in a Sorvall

RC-2B with an SS-34 rotor at 10,000 g for 20 min. The

protein concentration of the supernatant was determined

by the Bradford dye-binding assay (Bio-Rad, Hercules,

CA). The supernatant was saved for electroblotting. The

crude extract was subjected to 15% SDS-PAGE according

to Laemmli (Laemmli, 1970). After electrophoresis, gels

were equilibrated in transfer buffer (25 mM Tris-HCl,

pH 8.3, 150 mM glycine and 10% (w/v) methanol). The

separated proteins were transferred to an Immobilon

PVDF membrane (Millipore, Bedford, MA) in transfer

buffer at pH 8.3 for 1 h at 100 V. Membranes were blocked

for 2 h at room temperature in 5% nonfat dry milk powder

and then incubated with polyclonal antibodies from rabbit

as the primary antibodies against SPAP and TI. After

incubation, membranes were washed in Tris-buffered

saline with 0.05% Tween (TBST) three times, 10 min

each, then incubated with anti-rabbit alkaline phosphatase-

conjugated antibody, washed in TBST three times, 10 min

each, and developed using NBT (nitro blue tetrazolium)/

BCIP (5-bromo-4- chloro-3-indolyl-phosphate) (Sigma,

USA).

Phylogenic analysis of AP

Amino acid sequence alignment of AP after Genetics

Computer Inc. (GCG)/Pileup comparison was used for

phylogenic tree construction. The distances among entries

were calculated with neighbor-joining method (Thompson

et al., 1994). The internal support was evaluated by

bootstrap analyses. In parsimony analysis, each of 1,000

bootstrap replicates was analyzed with the heuristic search

option invoking one random addition replicate each, and

not invoking the retention of multiple parsimonious trees.

The phylogenic tree was drawn using NJ plot and redrawn

by the graphic software of CLUSTALX 1.81.

Proteinase activity

The general proteolytic activity of the enzyme was

monitored using denatured substrates such as casein,

hemoglobin, azoalbumin, and azocasein by the method

of Arnon (Arnon, 1970) with some modifications (Hou et

al., 2002). In a total final volume of 1 mL, 2% azocasein

(250 £gL) and 0.5-3 £gg of the enzyme were mixed and kept

at 37¢XC in a water bath for 30 min. The control assay was

performed without any enzyme in the reaction mixture.

The reaction was terminated by adding 0.5 mL of 10%

TCA and the absorbance at 340 nm of the supernatant was

measured.

ReSUlTS

Isolation and nucleotide sequence of aspartic

proteinase cDNA clone from sweet potato

storage roots

AP cDNA clones of sweet potato storage roots were

isolated by differential display. We have completed the

sequencing of one of the clones, which was named SPAP

(Gene Bank accession number: DQ903691). The open

reading frame in this cDNA encodes a pre-pro-protein

of 508 amino acids with a predicted molecular mass of

55,006 Da (pI 4.91). The SPAP gene shares 81% and 78%

homology on the level of nucleotides and amino acids,

respectively, with an aspartic proteinase cDNA of sweet

potato senescent leaves (SPAPSL) (Chen et al., 2004;

2008). SPAP amino acid sequence was different from

other AP sequences in both signal and propeptide portions.

Figure 1 shows a multiple alignment of the sweet potato

SPAP protein and other homologous plant precursor AP

proteins available in the GenBank (Figure 1). The deduced

amino acid sequence contains the conserved features of

plant aspartic proteinases, including the plant specific

insert (PSI).

A putative cleavage site on the hydrophobic N-terminal

signal peptide for targeting to ER was predicted between

Ser-24 and Glu-25 (Von Heijne, 1983; Nielsen et al.,

152

Botanical Studies, Vol. 50, 2009

Figu re 1. Multiple alignments of plant aspartic proteinase proteins. The sequences are aspartic proteinase SPAP (DQ903691)

from sweet potato storage roots, aspartic proteinase SPAPSL (AF216783) of sweet potato senescent leaves; LycoAP (L 46681) of

Lycopersicon esculentum; NepAP (AB045894) of Nepenthes alata; and GlyAP (AB070857) of Glycine max. The proteins were

aligned using the GCG program. The regions corresponding to the signal peptide (black dashed line), the prosegment (black

solid line) and the PSI (black dotted line) are marked. The catalytic aspartic acid residues are boxed. Black shading indicates the

same amino acid at that position among all sequences. Gray shading shows those amino acids with sim ilar side-chain proper-

ties. The numbers above all sequences stand for the positions of the amino acids within individual proteins corresponding to the

numbering system of Glycine max GlyAP, which is the longest among all sequences shown. T he numbers at the end of the right

hand side of each line stand for the cumulative total number of amino acids in each line of each preproprotein sequence.

HUANG et al. ¡X Aspartic proteinase: cloning and expression

153

1997). The two active site aspartic acid residues, one with

the Asp-Thr-Gly motif and the other Asp-Ser-Gly, are

consistent. Two putative N-glycosylation sites (270-273

NSST; 435-438 NVTF), one putative tyrosine kinase

phosphorylation site (73-81; KNYLDAQYY) (Pcgene,

Prosite program, Intelligenetics) (Richter, et al., 1998) and

one prokaryotic membrane lipoprotein lipid attachment

site (7-17; CASILLWVIAC) were predicted.

Phylogenic analysis of APs based on their

amino acid sequences

The phylogenic tree of SPAP together with amino acid

sequences of other 4 proteins were constructed (Figure 2).

Sweet potato aspartic proteinase SPAPSL was found in the

same subgroup, whereas SPAP was clustered in another

subgroup that included LycoSP and NepAP.

Copy numbers of aspartic proteinase

sequences in sweet potato

We performed Southern blot hybridization with EcoRI

(E), BamHI (B) and HindIII (H) digests of sweet potato

Tainong 57 DNA, using probe derived from 3¡¦-noncoding

sequence of the cDNAs to estimate the copy number of

the gene. Tainong 57, an elite sweet potato cultivar derived

from a cross between Tainong 27 and Nancy Hall, has a

hexaploid number of chromosome (2n=6x=90). The results

suggest that SPAP belongs to a small multigene family in

sweet potato (Figure 3).

expression of SPAP in E. coli

SDS-PAGE analysis of crude extracts from the

transformed E. coli (M15) showed high amounts of a

polypeptide with the molecular mass (ca. 55 kDa) of the

SPAP prepropeptide (Figure 4A). This polypeptide was

found as a soluble protein in the supernatant (Figure 4A,

lane 2), and was absent in protein extracts obtained from

E. coli transformed with pQE-30 vector (Figure 4A,

lane 1). The expressed protein was purified from crude

extracts as His-tagged SPAP (Figure 4A, lane 3). This

polypeptide was then analyzed by proteinase activity assay

according to Sundd et al (Sundd et al., 1998) (Figure 4B).

The expressed protein was able to degrade the substrate

azocasein indicating that SPAP has proteinase activity.

Aspartic proteinase mRNA levels were

developmentally regulated

The presence and amounts of different sweet potato

SPAP mRNAs were examined in various organs and

tissues by northern blot analysis. SPAP was obtained from

sweet potato tuberous roots. Figure 5A shows that SPAP

probe hybridized to mRNA species of approximately 1.5

kb. SPAP mRNA levels were the highest in the storage

roots, followed by that in sprouted roots and full expanded

green leaves; while was the lowest in sprout and vein.

Western blot analysis of aspartic proteinase

from sweet potato tissues

Western blot hybridization using SPAP polyclonal

antibody from rabbit antiserum was used for the gene

expression analysis of SPAP in crude extracts from

different sweet potato tissues (Figure 5B). SPAP levels

were the highest in the storage roots; followed by that in

sprouted. There were no signals in sprout roots, veins and

full expanded green leaves.

Figure 2. Phylogenetic analysis of aspartic proteinases based

on their amino acid sequences. The scale bar represents 0.02

units .

Fi gure 3. Sout her n blot de tec tion of aspar tic protei nase

genomic sequences. Samples (10 £gg) of genomic DNA from

sweet potato Tainong 57 le aves we re dige sted with EcoRI

(E), BamHI (B) and HindIII (H). The DNA fragments were

separated in 0.8% agarose gels, t ransferred to a Hybond-N

¡Ï

nylon membrane, a nd hybridized with PCR-labele d cDNA

probes. Molec ula r size marke rs we re £f DNA/HindIII frag-

ments. The experiments were done twice and a representative

one is shown.

154

Botanical Studies, Vol. 50, 2009

SPAP expression protein can degrade the

stored trypsin inhibitor proteins in vitro

It has been reported that inactivation of seed TI by

reductants improved protein digestibility (Huang et al.,

2005b). Formation of disulfide bonds of protein likely

functions to increase its structural stability and decrease its

water solubility, rendering it resistant against proteolysis.

Therefore, it would be interesting to study whether

reduction of stored TI by DTT or NTS can improve protein

digestibility in vitro.

The purified TI contained two bands (38 and 22 kDa)

on non-reducing SDS-PAGE under either protein-staining

or thiol labeling condition (lanes 1, 2 of Figure 6A and

6B, serving as controls). The existence of two forms of the

purified TI in controls could be explained by equilibrium

between TI38 and TI22 via interchain disulfide bond

formation, TI22 being a monomer and thermodynamically

favored. The evidence came from the reproducible

observations that when TI38 was cut from the gel and

stored in sample buffer at 4¢XC overnight and then run

non-reducing SDS-PAGE again, two bands (38 and 22

kDa) still could be observed; On the contrary, if TI22 was

treated similarity only one band (TI22) was detected (our

unpublished data).

SPAP protein could not degrade bands of native TI

(lanes 3 of Figure 6A). However, DTT could transform

TI38 into TI22 at room temperature. TI22 appeared

concomitantly with the disappearance of TI38 (lane 4,

F igure 5. Nor ther n and weste rn blot de tec tions of sweet

potato aspartic proteinase SPAP. A, Northern blot analysis.

Samples (10 £gg each) of total RNA were isolated from differ-

ent tissues of sweet potato a nd actin (AY905538) was utilized

as a n inte rnal c ont rol of m RNA from swe et pota to. Blots

were hybridized to £\-

32

P-labeled 3¡¦ specific cDNA probes; B,

West ern blots analysis. Ten £gg of crude extracted proteins

from sweet potato were analyzed by 15% (w/v) SDS/PAGE,

and then the gels were transferred ont o PVDF mem branes

that were probed with a 1:1000 (v/v) dilution of rabbit anti-

bodies raised against SPAP usi ng goat-anti ra bbit alkali ne

phosphatase as the second antibody. Lane 1: storage roots,

lane 2: sprout, lane 3: veins, lane 4: sprouted roots, and lane

5: fully expanded green leaves. The experiments were done

twice and a representative one is shown.

Figure 4. Purified recombinant sweet potato aspartic pro-

teinase SPAP. A, 10% SDS-PAGE analysis. Crude ext racts (5

£gg protein) from E . coli (M15) transformed with pQE30 (lane

1) or with pQE30-SPAP (lane 2) were analyzed by 10% (w/v)

SDS-PAGE, and then the gels were stained with Coomassie

brilliant blue G-250. Molecular masses of standard proteins

were indicated at the left of the figure. His-tagged SPAP was

purified by Ni

2+

-chelated affinity chromatography (lane 3);

B, Proteinase activity analysis. The experiments were done

twice and a representative one is shown. "M" indicated the

see Blue

TM

pre-stained markers for SDS-PAGE.

HUANG et al. ¡X Aspartic proteinase: cloning and expression

155

Figure 6A). Scanning the mBBr-TI bands with a laser

densitometer revealed that TI22 increased after DTT

treatment (lane 4, Figure 6B). In the presence of DTT and

expressed SPAP protein, TI22 was degraded completely

(lane 5, Figure 6A). At the same time, the mBBr-protein

band of TI22 disappeared (lane 5, Figure 6B).

When NTS (NADP/thioredoxin system) was used

to reduce TI, SPAP protein could not degrade bands of

reduced TI (data not shown). The results indicated that

SPAP protein could degrade TI reduced by DTT that cause

drastic 3-D conformational changes of TI.

DISCUSSION

We have presented a nucleotide sequence of an

aspartic proteinase cDNA clone from sweet potato. The

protein encoded by the open reading frame contains 508

amino acids. Plant SPAP cDNAs have been detected and

isolated from many different plant species. SPAP amino

acid sequence was different from other AP sequences in

both signal and propeptide portions. The deduced amino

acid sequence contains the conserved features of plant

aspartic proteinases, including the plant specific insert

(PSI). According to the cDNA sequence analysis SWAP,

SPAP protein like most plant APs has an internal domain

(PSI) with a high homology to saposin-like proteins

(SAPLIPs) type B. This protein family has been associated

with antitumoral and antimicrobial activity due to their

membrane leakage activity. The PSI presence in mature

APs could explain bifunctional activity (proteolytic and

antimicrobial) (Guevara et al., 2005).

The functions of the PSI are still unclear, however, an

important role in vacuolar targeting of plant AP precursors

has been proposed based on its possible direct interaction

with lipid bilayers (Munford et al., 1995; Vaccaro et al.,

1999). Thus, this saposin-like domain in plant APs may

be responsible for bringing AP precursors into contact

with membranes or membrane-bound receptor proteins

mediating the sorting of enzyme precursors during Golgi-

mediated intracellular transport to the vacuoles (Egas et

al., 2000).

SPAP protein could degrade TI22 reduced by DTT.

When NTS was used to reduce TI, SPAP protein could

not degrade bands of reduced TI (data not shown). In our

previous paper, the NTS could reduce TI proteins in vitro

rendering them to be degraded by suitable proteinases

such as SPCPRPP protein (Huang et al., 2005b). These

results suggest that CPR is responsible for initiation of

degradation and re-mobilization of stored 22 kDa TI

during sprouting of SP storage roots after the reduction

of 22 kDa TI by the NTS (step 1, Figure 7). And SPAP

protein may be responsible for the later step of degradation

(step 2, Figure 7).

In castor bean aspartic endopeptidase cannot directly

convert pro2S albumin into the mature form, but it may

play a role in trimming the C-terminal propeptides from

the subunits that are produced by the action of the vacuolar

processing enzyme (Hiraiwa et al., 1997). In citrus leaf

extracts, an AP has been implicated in the proteolysis of

the photosynthetic enzyme ribulose-1, 5-bisphosphate

Figure 6. Reduction of t rypsin inhibitor from sweet potato

storage root s by DTT and digestion with the re combinant

SPAP protein. (A) Protei n st ainings of TI with Coomassie

brilliant blue G250 were performed on 15% SDS-PAGE gels;

(B) T he fluorescence of samples (thiol-labeling) after reduc-

tion was detected on 15% mBBr-containing SDS-PAGE gels.

L ane 1, t ry psin inh ibitor at 0oC for 24 h; lane 2, tr ypsin

inhibitors incubated at 37oC for 24 h; lane 3, trypsin inhibi-

tors and 1 £gg SPAP were incubated at 37oC for 24 h; lane 4,

t rypsin inhibitor plus 2 m M DT T was incubated at 37oC for

24 h; lane 5, trypsin inhibitor, 2 m M DTT and 1 £gg SPAP

were incubated at 37oC for 24 h. The experiments were done

t wice and a representative one is shown. Each lane contained

10 £gg pur if ied SPT I. " M" indi cat ed the see Blue

TM

pre-

stained markers for SDS-PAGE.

Figu re 7. A proposed degradation steps of trypsin inhibitor

from sweet potato storage roots.

156

Botanical Studies, Vol. 50, 2009

carboxylase/oxygenase which plays a significant role

as a nitrogen source during the growth of new organs

(Garciamartinez et al., 1986). Participation of plant APs

in storage protein degradation during the mobilization of

reserve proteins in seed germination has been proposed

for rice and wheat. In rice seeds AP could be involved

in the hydrolysis of £^-globulin during the initial stage of

germination because both proteins are similarly distributed

in the seeds (Doi et al., 1980). In wheat seeds AP could

hydrolyse the storage protein, gliadin, in vitro (Belozersky

et al., 1989).

Therefore, our data provide evidence for the first time

that stored TI could be degraded initially by a specific

cysteine proteinase and then by AP more efficiently in

vitro and possibly in vivo.

lITeRATURe CITeD

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a s parti c prote ina se , oryza si n, expre ss ed du ring s eed

ripening and germination, has a gene organization distinct

from those of animal and microbial as partic proteinases.

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Barrett, D.R. 1998. Proteolytic enzymes: nomenclature

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Proteolytic Enzymes, Academic Press, London, pp. 1-20.

Belozersky, M.A., S.T. Sarbakanova, and Y.E. Dunaevsky. 1989.

Aspartic proteinase from wheat seeds - isolation, properties

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